Modulation of HIF1α and HIF2α expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of HIF1α and/or HIF2α. The compositions comprise oligonucleotides, targeted to nucleic acid encoding HIF1α and HIF2α. Methods of using these compounds for modulation of HIF1α and/or HIF2α expression and for diagnosis and treatment of disease associated with expression of HIF1α and/or HIF2α are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 10/304,126filed Nov. 23, 2002 now U.S. Pat. No. 7,144,999.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of HIF1α (HIF1α) and hypoxia-inducible factor 2 alpha(HIF2α). In particular, this invention relates to compounds,particularly oligonucleotide compounds, which, in preferred embodiments,hybridize with nucleic acid molecules encoding HIF1α and HIF2α. Suchcompounds are shown herein to modulate the expression of HIF1α andHIF2α.

BACKGROUND OF THE INVENTION

Oxygen homeostasis is an essential cellular and systemic function;hypoxia leads to metabolic demise, but this must be balanced by the riskof oxidative damage to cellular lipids, nucleic acids, and proteinsresulting from hyperoxia. As a result, cellular and systemic oxygenconcentrations are tightly regulated via response pathways that affectthe activity and expression of a multitude of cellular proteins. Thisbalance is disrupted in heart disease, cancer, cerebrovascular disease,and chronic obstructive pulmonary disease (Semenza, Genes Dev., 2000,14, 1983–1991) (Semenza, G., 2001, Trends Mol. Med., 7, 345–350. Cellsare typically cultured in the laboratory at an ambient oxygenconcentration of 21%, but cells in the human body are exposed to muchlower oxygen concentrations ranging from 16% in the lungs to less than6% in most other organs of the body often significantly less in tumors.Semenza G., 2001, Trends Mol. Med., 7, 345–350.

Solid tumor growth depends on a continuous supply of oxygen andnutrients through neovascularization (angiogenesis). Tumors often becomehypoxic, often because new blood vessels are aberrant and have poorblood flow. Cancer cells make adaptive changes that allow them toproliferate even at hypoxia. These changes include an increase inglycolysis and an increase in production of angiogenic factors. Hypoxiain tumors is associated with resistance to radio- and chemotherapy, andthus is an indicator of poor survival.

The transcriptional complex, hypoxia inducible factor (HIF), is a keyregulator of oxygen homeostasis. Hypoxia induces the expression of genesparticipating in many cellular and physiological processes, includingoxygen transport and iron metabolism, erythropoiesis, angiogenesis,glycolysis and glucose uptake, transcription, metabolism, pH regulation,growth-factor signaling, response to stress and cell adhesion. Thesegene products participate in either increasing oxygen delivery tohypoxic tissues or activating an alternative metabolic pathway(glycolysis) which does not require oxygen. Hypoxia-induced pathways, inaddition to being required for normal cellular processes, can also aidtumor growth by allowing or aiding angiogenesis, immortalization,genetic instability, tissue invasion and metastasis (Harris, Nat. Rev.Cancer, 2002, 2, 38–47; Maxwell et al., Curr. Opin. Genet. Dev., 2001,11, 293–299).

HIF is a heterodimer composed of an alpha subunit complexed with a betasubunit, both of which are basic helix-loop-helix transcription factors.The beta subunit of HIF is a constitutive nuclear protein. The alphasubunit is the regulatory subunit specific to the oxygen responsepathway, and can be one of three subunits, HIF1α, 2 alpha or 3 alpha(HIF-1α, HIF-2α and HIF-3α, respectively) (Maxwell et al., Curr. Opin.Genet. Dev., 2001, 11, 293–299; Safran and Kaelin, J. Clin. Invest.,2003, 111, 779–783).

The transcription factor hypoxia-inducible factor 1 (HIF-1) plays anessential role in homeostatic responses to hypoxia by binding to the DNAsequence 5′-TACGTGCT-3′ and activating the transcription of dozens ofgenes in vivo under hypoxic conditions (Wang and Semenza, J. Biol.Chem., 1995, 270, 1230–1237). These gene products participate in eitherincreasing oxygen delivery to hypoxic tissues or activating analternative metabolic pathway (glycolysis) which does not requireoxygen. This list includes: aldolase C, enolase 1, glucose transporter1, glucose transporter 3, glyceraldehyde-3-phosphate dehydrogenase,hexokinase 1, hexokinase 2, insulin-like growth factor-2 (IGF-2), IGFbinding protein 1, IGF binding protein 3, lactate dehydrogenase A,phosphoglycerate kinase 1, pyruvate kinase M, p21, transforming growthfactor B3, ceruloplasmin, erythropoietin, transferrin, transferrinreceptor, a1b-adrenergic receptor, adrenomedullin, endothelin-1, hemeoxygenase 1, nitric oxide synthase 2, plasminogen activator inhibitor 1,vascular endothelial growth factor (VEGF), VEGF receptor FTL-1, and p35(Semenza, Genes Dev., 2000, 14, 1983–1991). Expression of HIF1α is alsosensitive to oxygen concentration: increased levels of protein aredetected in cells exposed to 1% oxygen and these decay rapidly uponreturn of the cells to 20% oxygen (Wang et al., Proc. Natl. Acad. Sci.U.S.A., 1995, 92, 5510–5514).

Hypoxia-inducible factor-1 alpha is a heterodimer composed of a 120 kDaalpha subunit complexed with a 91 to 94 kDa beta subunit, both of whichcontain a basic helix-loop-helix (Wang and Semenza, J. Biol. Chem.,1995, 270, 1230–1237). The gene encoding hypoxia-inducible factor-1alpha (HIF1α, also called HIF-1 alpha, HIF1A, HIF-1A, HIF1-A, and MOP1)was cloned in 1995 (Wang et al., Proc. Natl. Acad. Sci. U.S.A., 1995,92, 5510–5514). A nucleic acid sequence encoding HIF1α is disclosed andclaimed in U.S. Pat. No. 5,882,914, as are expression vectors expressingthe recombinant DNA, and host cells containing said vectors (Semenza,1999).

HIF1α expression and HIF-1 transcriptional activity are preciselyregulated by cellular oxygen concentration. The beta subunit is aconstitutive nuclear protein, while the alpha subunit is the regulatorysubunit. HIF1α mRNA is expressed at low levels in tissue culture cells,but it is markedly induced by hypoxia or ischemia in vivo (Yu et al., J.Clin. Invest., 1999, 103, 691–696). HIF1α protein is negativelyregulated in non-hypoxic cells by ubiquitination and proteasomaldegradation (Huang et al., Proc. Natl. Acad. Sci. U.S.A., 1998, 95,7987–7992). Under hypoxic conditions, the degradation pathway isinhibited, HIF1α protein levels increase dramatically, and the fractionthat is ubiquitinated decreases. HIF1α then translocates to the nucleusand dimerizes with a beta subunit (Sutter et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 4748–4753).

A natural antisense transcript that is complementary to the 3′untranslated region of HIF1α mRNA has been discovered and is named“aHIF” (Thrash-Bingham and Tartof, J. Natl. Cancer Inst., 1999, 91,143–151). This is the first case of overexpression of a naturalantisense transcript exclusively associated with a specific humanmalignant disease. aHIF is specifically overexpressed in nonpapillaryclear-cell renal carcinoma under both normoxic and hypoxic conditions,but not in papillary renal carcinoma. Although aHIF is not furtherinduced by hypoxia in nonpapillary disease, it can be induced inlymphocytes where there is a concomitant decrease in HIF1α mRNA.

HIF1α plays an important role in promoting tumor progression and isoverexpressed in common human cancers, including breast, colon, lung,and prostate carcinoma. Overexpression of HIFs is sometimes observed incancers, such as clear cell renal cell carcinoma, even at normoxia.Mutations that inactivate tumor suppressor genes or activate oncogeneshave, as one of their consequences, upregulation of HIF1α activity,either through an increase in HIF1α protein expression, HIF1αtranscriptional activity, or both (Semenza, Pediatr. Res., 2001, 49,614–617).

Until a tumor establishes a blood supply, the hypoxic conditions limittumor growth. Subsequent increases in HIF1α activity result in increasedexpression of target genes such as vascular endothelial growth factor(VEGF). VEGF expression is essential for vascularization and theestablishment of angiogenesis in most solid tumors (Iyer et al., GenesDev., 1998, 12, 149–162). A significant association betweenhypoxia-inducible factor-1 alpha, VEGF overexpression and tumor grade isalso seen in human glioblastoma multiforme, the highest grade glioma inwhich mean patient survival time is less than one year. The rapidlyproliferating tumor outgrows its blood supply, resulting in extensivenecrosis, and these regions express high levels of HIF1α protein andVEGF mRNA, suggesting a response of the tumor to hypoxia (Zagzag et al.,Cancer, 2000, 88, 2606–2618).

The action of the von Hippel-Landau (VHL) tumor suppressor gene productis implicated in hypoxic gene regulation, in both normal and diseasedcells. Individuals with VHL disease are predisposed to renal cysts,clear cell renal carcinoma, phaeochromocytoma, haemangioblastomas of thecentral nervous system, angiomas of the retina, islet cell tumors of thepancreas, and endolymphatic sac tumors (Pugh and Ratcliffe, Semin.Cancer. Biol., 2003, 13, 83–89). The VHL gene product participates inubiquitin-mediated proteolysis by acting as the recognition component ofthe E3-ubiquitin ligase complex involved in the degradation ofhypoxia-inducible factor alpha subunits (Cockman et al., J. Biol. Chem.,2000, 275, 25733–25741; Ohh et al., Nat. Cell Biol., 2000, 2, 423–427).In normal cells, VHL/HIF complexes form and target HIF alpha subunitsfor destruction (Maxwell et al., Nature, 1999, 399, 271–275). This isproposed to occur through hydroxylation of the oxygen-dependent domainof HIF2α and subsequent recognition by the VHL gene product, asrecognition of a homologous oxygen-dependent domain is the mechanism bywhich the VHL protein recognizes HIF1α (Maxwell et al., Nature, 1999,399, 271–275). HIF2α is in fact hydroxylated by the enzyme prolyl4-hydroxylases in vitro (Hirsila et al., J. Biol. Chem., 2003).

The p53 tumor suppressor also targets HIF1α for degradation by theproteasome. Loss of p53 activity occurs in the majority of human cancersand indicates that amplification of normal HIF1α levels contributes tothe angiogenic switch during tumorigenesis (Ravi et al., Genes Dev.,2000, 14, 34–44).

A mouse model of pulmonary hypertension has shown that local inhibitionof HIF1α activity in the lung might represent a therapeutic strategy fortreating or preventing pulmonary hypertension in at risk individuals. Inpulmonary hypertension hypoxia-induced vascular remodeling leads todecreased blood flow, which leads to progressive right heart failure anddeath. This hypoxia-induced vascular remodeling is markedly impaired inmice that are partially HIF1α deficient (Yu et al., J. Clin. Invest.,1999, 103, 691–696). Decreased vascular density and retarded solid tumorgrowth is also seen in mouse embryonic stem cells which are deficientfor HIF1α (Ryan et al., Embo J, 1998, 17, 3005–3015).

During hypoxia, cells shift to a glycolytic metabolic mode for theirenergetic needs and HIF1α is known to upregulate the expression of manyglycolytic genes. HIF1α may play a pivotal role in the Warburg effect intumors, a paradoxical situation in which tumor cells growing undernormoxic conditions show elevated glycolytic rates, which enhances tumorgrowth and expansion. HIF1α mediates the expression of6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3, a gene whoseprotein product maintains levels of the key regulator of glycolyticflux, fructose-2,6-bisphosphate (Minchenko et al., J. Biol. Chem., 2001,14, 14).

Currently, there are no known therapeutic agents which effectivelyinhibit the synthesis of HIF1α and to date, investigative strategiesaimed at modulating HIF1α function have involved the use of antisenseexpression vectors and oligonucleotides. These studies have served todefine the involvement of HIF1α in disease progression and to identifynovel roles of HIF1α in vivo including unique roles for HIF1α as atranscription factor under non-hypoxic conditions and as an inhibitor ofgene expression.

Gene transfer of an antisense HIF1α plasmid has been shown to enhancethe efficacy of cancer immunotherapy. Antisense therapy was shown toslow, but not eradicate, the growth of EL-4 tumors established in mice.In addition, endogenously expression of HIF1α was almost completelyinhibited in these tumors. When antisense therapy was combined withT-cell costimulator B7-1 immunotherapy, the tumors completely andrapidly regressed within 1 week. Furthermore, when these tumor-free micewere rechallenged with EL-4 cells, no tumors emerged, indicating thatsystemic antitumor immunity had been achieved (Sun et al., Gene Ther.,2001, 8, 638–645).

Activation of HIF1α is thought to aggravate heart failure byupregulation of cardiac ET-1, a gene product involved in heart failureand whose inhibition improves the survival rate of rats with heartfailure. In a failing heart, a metabolic switch occurs, and HIF1αactivates the expression of glycolytic enzymes as compensation forimpaired b-oxidation of fatty acid. Another consequence of increasedHIF1α activity is that in rat cardiomyocytes, HIF1α was shown to bind tothe 5′-promoter region of the ET-1 gene and increase ET-1 expression. Invitro, an antisense oligonucleotide targeted to hypoxia-induciblefactor-1 alpha largely inhibited the increased gene expression of ET-1,confirming the role of HIF1α in heart failure (Kakinuma et al.,Circulation, 2001, 103, 2387–2394). This antisense oligonucleotide iscomprised of 20 nucleotides and targets bases 11 to 31 of the rat HIF1αwith GenBank accession number AF_(—)057308 incorporated herein byreference.

Preeclampsia is a disorder of unknown etiology that is the leading causeof fetal and maternal morbidity and mortality. Defective downregulationof HIF1α may play a major role in the pathogenesis of preeclampsia. Formost of the first trimester, the human fetus develops under hypoxicconditions but at 10–12 weeks the intervillous space opens, the fetus isexposed to maternal blood and at this stage the trophoblast cells invadethe maternal decidua. The switch of the trophoblasts from aproliferative to an invasive phenotype is controlled by cellular oxygenconcentration. The proliferative, non-invasive trophoblast phenotypeappears to be maintained by HIF1α mediated expression of TGFbeta3because treatment of human villous explants with an antisenseoligonucleotide against HIF1α or TGF beta 3 induces invasion underhypoxic conditions. In this case the HIF1α antisense oligonucleotide wascomprised of phosphorothioate oligonucleotides, 16 nucleotides inlength, and targeted to the AUG codon (Caniggia et al., J. Clin.Invest., 2000, 105, 577–587.; Caniggia et al., Placenta, 2000, 21 SupplA, S25–30).

The human intestinal trefoil factor (ITF) gene product protects theepithelial barrier during episodes of intestinal hypoxia. The ITF genepromoter bears a bindingsite for hypoxia-inducible factor-1 alpha, andthe function of HIF1α as a transcription factor for ITF was confirmed invitro. T84 colonic epithelial cells were treated with a phosphorothioateantisense oligonucleotide, 15 nucleotides in length and targeted to theAUG codon of HIF1α and this resulted in a loss of ITF hypoxiainducibility (Furuta et al., J. Exp. Med., 2001, 193, 1027–1034).

Human epidemiological and animal studies have associated inhalation ofnickel dusts with an increased incidence of pulmonary fibrosis. Nickeltranscriptionally activates plasminogen activator inhibitor (PAI-1), aninhibitor of fibrinolysis, through the HIF1α signaling pathway. This wasevidenced by decreases in PAI-1 mRNA levels when human airway epithelialcells were treated with an antisense oligonucleotide directed againstHIF1α identical to the one used in the preeclampsia study discussedabove. These data may be critical for understanding the pathology ofpulmonary fibrosis and other diseases associated with nickel exposure(Andrew et al., Am J Physiol Lung Cell Mol Physiol, 2001, 281,L607–615).

HIF1α is constitutively expressed in cerebral neurons under normoxicconditions. A second dimerization partner for HIF1α is ARNT2, a cerebraltranslocator homologous to hypoxia-inducible factor-1 beta. One splicevariant of HIF1α found in rat neurons dimerizes with ARNT2 more avidlythan it does with HIF1b, and the resulting hypoxia-inducible factor-1alpha-ARNT2 heterodimer does not recognize the HIF1α binding site of theerythropoietin gene. This suggests that transcription of a different setof genes is controlled by the hypoxia-inducible factor-1 alpha-ARNT2heterodimer controls in neurons under nonhypoxic conditions than thehypoxia-inducible factor-1 alpha-HIF1α heterodimer controls underhypoxic conditions. This was evidenced by antisense oligonucleotidedownregulation of HIF1α expression in which the antisenseoligonucleotide consisted of 16 phosphorothioate nucleotides targeted tobases 38 to 54 of the rat hypoxia-inducible factor-1 with GenBankaccession number AF_(—)057308 (Drutel et al., Eur. J. Neurosci., 2000,12, 3701–3708).

A role for HIF1α in mediating a down-regulatory pathway was recentlydiscovered using antisense oligonucleotide depletion ofhypoxia-inducible factor-1 alpha. The peroxisome proliferator-activatedreceptors (PPARS) are a nuclear hormone-binding proteins that regulatetranscriptional activities. Ligands which bind the PPAR-gamma isoformman amplify or inhibit the expression of inflammation-related geneproducts and may regulate the duration of inflammatory response. Hypoxiaelicits a down-regulation of PPAR-gamma in intestinal epithelial cellswhich is effected through a binding site for HIF1α on the antisensestrand of the PPAR-gamma gene. The expression of PPAR-gamma wasupregulated in hypoxic cells when treated with an antisenseoligonucleotide targeted to HIF1α identical to the one used in thepreeclampsia study discussed above (Narravula and Colgan, J. Immunol.,2001, 166, 7543–7548).

The gene encoding hypoxia-inducible factor 2 alpha (HIF2α; also calledHIF-2 alpha, endothelial PAS domain protein 1, EPAS1, MOP2,hypoxia-inducible factor 2, HIF-related factor, HRF, HIF1 alpha-likefactor, HLF) was initially identified as a transcription factorexpressed in endothelial cells (Ema et al., Proc. Natl. Acad. Sci.U.S.A., 1997, 94, 4273–4278; Flamme et al., Mech. Dev., 1997, 63, 51–60;Hogenesch et al., J. Biol. Chem., 1997, 272, 8581–8593; Tian et al.,Genes Dev., 1997, 11, 72–82). A nucleic acid sequence encoding humanHIF2α is disclosed and claimed in U.S. Pat. No. 5,695,963 (McKnight etal., 1997).

HIF2α mRNA is primarily expressed in highly vascularized adult tissues,such as lung, heart and liver, and in the placenta and endothelial cellsof the embryonic and adult mouse (Hogenesch et al., J. Biol. Chem.,1997, 272, 8581–8593). Comparison of normal human tissues and cancersreveals that HIF2α protein is not detectable in normal tissue, but iseasily visualized in malignant tissues (Talks et al., Am. J. Pathol.,2000, 157, 411–421). The requirement for expression of HIF2α indevelopment is demonstrated by the abnormalities observed in HIF2α genedeficient mouse embryos, which include the disruption of catecholaminehomeostasis and lack of protection against heart failure observed (Tianet al., Genes Dev., 1998, 12, 3320–3324). Targeted disruption of theHIF2α gene and generation of embryos deficient for HIF2α is disclosed inthe PCT publication WO 02/086497 (Compernolle et al., 2002). Thispublication also discloses antisense oligodeoxyribonucleotides for usein inhibiting HIF2α expression targeted to the translation initiationcodon of HIF2α (Compernolle et al., 2002).

HIF2α expression and HIF transcriptional activity are preciselyregulated by cellular oxygen concentration. Whereas changes in oxygenlevels do not affect HIF1-beta protein levels, the abundance of thealpha subunits is markedly increased upon exposure of cells to hypoxia,primarily due to stabilization of the alpha subunit protein (Safran andKaelin, J. Clin. Invest., 2003, 111, 779–783). HIF2α mRNA and protein isexpressed at low levels in tissue culture cells, but protein expressionis markedly induced by exposure to 1% oxygen, a hypoxic state (Wieseneret al., Blood, 1998, 92, 2260–2268). The hypoxia-inducible factor 2alpha/hypoxia-inducible factor 1 beta heterodimer protein binds to thehypoxic response element, which contains the core recognition sequence5′-TACGTG-3′ and is found in the cis-regulatory regions ofhypoxia-regulated genes (Ema et al., Proc. Natl. Acad. Sci. U.S.A.,1997, 94, 4273–4278; Hogenesch et al., J. Biol. Chem., 1997, 272,8581–8593). Binding of the heterodimer to the HRE induces geneexpression. Upon return to normoxic conditions, HIF2α protein is rapidlydegraded (Wiesener et al., Blood, 1998, 92, 2260–2268).

The mitogen-activated protein kinase (MAPK) pathway is critical forHIF2α activation. Inhibition of a dual specificity protein kinase thatdirectly phosphorylates MAPK prevents HIF2α trans-activation duringhypoxia (Conrad 1999; Conrad, 2001). However, the inhibitor does notprevent HIF2α phosphorylation, thus, while the MAPK pathway regulatesthe activity of hypoxia-inducible factor 2 alpha, it does not directlyphosphorylate the protein (Conrad et al., Comp. Biochem. Physiol. B.Biochem. Mol Biol., 2001, 128, 187–204; Conrad et al., J. Biol. Chem.,1999, 274, 33709–33713). The Src family kinase pathway is alsoimplicated in regulation of hypoxia-inducible factor 2 alpha. A specificinhibitor of the Src family of kinases abolishes the hypoxia-inducedexpression of HIF2α mRNA in human lung adenocarcinoma cells (Sato etal., Am. J. Respir. Cell Mol. Biol., 2002, 26, 127–134).

The maintenance of oxygen homeostasis, in addition to being required inphysiological development, is also required in tumor growth. Tumor cellsexperience hypoxia because blood circulates poorly through the aberrantblood vessel that tumors establish. Although hypoxia is toxic to cancercells, they survive as a result of genetic and adaptive changes thatallow them to thrive in a hypoxic environment. One such adaptation is anincrease in the expression of the angiogenic growth factor namedvascular endothelial growth factor (VEGF). VEGF is a key angiogenicfactor secreted by cancer cells, as well as normal cells, in response tohypoxia (Harris, Nat. Rev. Cancer, 2002, 2, 38–47; Maxwell et al., Curr.Opin. Genet. Dev., 2001, 11, 293–299).

Hemangioblastomas, the most frequent manifestation of VHL genemutations, exhibit overexpression of VEGF mRNA in their associatedstromal cells. The VEGF mRNA overexpression is highly correlated withelevated expression of HIF2α mRNA. This finding suggests a relationshipbetween loss of function of the VHL gene, and transcriptional activationof the VEGF gene, possibly through HIF2α activity in VEGF-dependentvascular growth (Flamme et al., Am. J. Pathol., 1998, 153, 25–29).

The tumor suppressive activity of the VHL gene product can be overriddenby the activation of HIF target genes in human renal carcinoma cells invivo. VHL gene product mutants lose the ability to target HIF forubiquitin-mediated destruction, suggesting that down regulation of HIFand VHL tumor suppressor function are intimately linked (Kondo et al.,Cancer Cell, 2002, 1, 237–246). In contrast to human renal cellcarcinoma, the product of the tuberous sclerosis complex-2 (Tsc-2) gene,product rather than VHL gene, is the primary target for rodent renalcell carcinoma (Liu et al., Cancer Res., 2003, 63, 2675–2680). Rat RCCcells lacking Tsc-2 function exhibit stabilization of HIF2α protein andupregulation of VEGF, and were highly vascularized (Liu et al., CancerRes., 2003, 63, 2675–2680).

A link between elevated HIF2α activity and angiogenesis has also beendemonstrated by experiments that show how HIF activity regulates VEGFexpression. Normal human kidney cells typically have low levels ofhypoxia-inducible factor 2 alpha, but upon introduction of a vectorencoding HIF2α into these cells, VEGF mRNA and protein levels increasesignificantly (Xia et al., Cancer, 2001, 91, 1429–1436). When HIF2α wasinhibited, VEGF expression was significantly decreased, thusdemonstrating a direct link between HIF2αactivity and VEGF expression(Xia et al., Cancer, 2001, 91, 1429–1436). Similarly, a dose-dependentincrease in VEGF mRNA is observed when human umbilical vein cells aretransduced with a virus encoding HIF2α (Maemura et al., J. Biol. Chem.,1999, 274, 31565–31570). Expression of a mutated HIF2α that lacks atransactivation domain inhibits the induction of VEGF mRNA duringhypoxia, a finding that further suggests that HIF2α is an importantregulator of VEGF expression (Maemura et al., J. Biol. Chem., 1999, 274,31565–31570).

A correlation between HIF activity and VEGF expression is also observedin malignant cells and tissues. HIF2α can be readily detected in renalcell carcinoma (RCC) cell lines in the absence of a vector encodingHIF2α (Xia et al., Cancer, 2001, 91, 1429–1436). Significant increasesin HIF2α and VEGF mRNA in renal cell carcinoma tissue samples, comparedto normal tissue, suggest that abnormal activation of HIF2α may beinvolved in the angiogenesis of RCC (Xia et al., Cancer, 2001, 91,1429–1436).

In addition to RCC, the expression of HIF2α in other malignancies hasalso been reported. HIF2α is expressed at the levels of mRNA and proteinin human bladder cancers, especially in those with an invasive phenotype(Xia et al., Urology, 2002, 59, 774–778). Another example ofoverexpression of HIF2α is seen in squamous cell head-and-neck cancer(SCHNC). Higher levels of HIF2α were associated with locally aggressivebehavior of SCHNC, as well as intensification of angiogenesis(Koukourakis et al., Int. J. Radiat. Oncol. Biol. Phys., 2002, 53,1192–1202). These findings also demonstrated a link betweenoverexpression of HIF2α and resistance to chemotherapy. Yet anothercorrelation between overexpression of HIF2α and cancer is seen inmalignant pheochromocytomas, which exhibit a higher level of HIF2α andan induced VEGF pathway, when compared to benign counterparts (Favier etal., Am. J. Pathol., 2002, 161, 1235–1246). HIF2α overexpression is alsoa common event in non-small-cell lung cancer (NSCLC) and is related tothe up-regulation of multiple angiogenic factors and overexpression ofangiogenic receptors by cancer cells. HIF2α overexpression in NSCLC isan indicator of poor prognosis (Giatromanolaki et al., Br. J. Cancer,2001, 85, 881–890). Taken together, these studies demonstrate thatelevated HIF2α confers aggressive tumor behavior, and that targeting theHIF pathway may aid the treatment of several different types of cancers.

Overexpression of HIF2α has also been observed in several cancer celllines in addition to RCC cell lines. Elevated levels of HIF2α mRNA andprotein are seen in human lung adenocarcinoma cells, and exposure ofthese cells to hypoxia further increases HIF2α expression (Sato et al.,Am. J. Respir. Cell Mol. Biol., 2002, 26, 127–134). Furthermore, thehypoxia response element plays a role in constitutively upregulating anisoform of VEGF in cancer cell lines under normoxic conditions. The HRElocated within a cell type-specific enhancer element in glioblastomacells participates in the up-regulation of VEGF expression throughenhanced binding of HIF2α to the HRE (Liang et al., J. Biol. Chem.,2002, 277, 20087–20094). A truncated version of HIF2α that can bind tohypoxia-inducible factor 1 beta, but not to the HRE, was unable totransactivate the VEGF promoter (Liang et al., J. Biol. Chem., 2002,277, 20087–20094). This further demonstrates the capability of cancercells to combat hypoxic conditions by enhancing expression of factorsrequired for vascularization and angiogenesis.

Short interfering RNAs (siRNAs) have been used to specifically inhibitthe expression of HIF1α and HIF2α in human breast and renal carcinomacell lines and in a human endothelial cell line. SiRNA duplexes withdTdT overhangs at both ends were designed to target nucleotides1521–1541 and 1510–1530 of the HIF1α mRNA sequence (NM001530) andnucleotides 1260–1280 and 328–348 of the HIF2α sequence (NM001430). Itwas found that in the breast carcinoma and endothelial cell lines, geneexpression and cell migration patterns were critically dependent onHIF1α but not hypoxia-inducible factor-2 alpha, but critically dependenton HIF2α in the renal carcinoma cells. Sowter et al., 2003, Cancer Res.,63, 6130–6134.

Defective downregulation of HIF2α may play a major role in thepathogenesis of preeclampsia. HIF2α protein levels are increased duringearly development, as expected in a hypoxic environment, and thendecrease significantly with gestational age (Rajakumar and Conrad, Biol.Reprod., 2000, 63, 559–569). However, HIF2α protein expression issignificantly increased in preeclamptic relative to normal termplacentas (Rajakumar et al., Biol. Reprod., 2001, 64, 499–506). Thisresult suggests that failure to down-regulate HIF2α protein expressionduring early pregnancy could prevent the switch of the trophoblast to aninvasive phenotype and ultimately lead to preeclampsia (Rajakumar etal., Biol. Reprod., 2001, 64, 499–506).

Overexpression of hypoxia-inducible factor 2 alpha, as well ashypoxia-inducible factor 1, has been observed in the inflammatory boweldiseases Crohn's disease and ulcerative colitis (Giatromanolaki et al.,J. Clin. Pathol., 2003, 56, 209–213). However, VEGF expression was weakin ulcerative colitis samples, and absent in Crohn's disease samples.The discordant expression of VEGF and HIF2α may lead to a reducedability of a tissue to produce or respond to VEGF, which may in turnlead to reduced endothelial and epithelial cell viability(Giatromanolaki et al., J. Clin. Pathol., 2003, 56, 209–213).

In addition to participating in adaptive changes in response to hypoxia,HIF2α may also function in an inflammatory response in cardiac myocytes.In cultured cardiac myocytes, interleukin-1 beta (IL-1beta)significantly increased both HIF2α mRNA and protein levels (Tanaka etal., J. Mol. Cell Cardiol., 2002, 34, 739–748). Transduction of cardiacmyocytes with adenovirus expressing HIF2α dramatically increased thelevels of adrenomedullin (AM) mRNA, which is also upregulated byIL-lbeta (Tanaka et al., J. Mol. Cell Cardiol., 2002, 34, 739–748).Since IL-1 beta has been implicated in the pathogenesis of heartfailure, and AM is known to improve cardiac function during heartfailure, these results suggest that HIF2α plays a role in the adaptationof the cardiac myocytes during heart failure (Tanaka et al., J. Mol.Cell Cardiol., 2002, 34, 739–748).

Disclosed and claimed in the PCT publication WO 00/09657 is a method ofinhibiting angiogenesis in a mammal through administration of a compoundwhich inhibits the binding of human HIF2α protein to the DNA regulatoryelement of an angiogenic factor, wherein the compound can be anantisense nucleic acid molecule complementary to all or part of the mRNAencoding human HIF2α (Lee et al., 2000). This publication also disclosesa nucleic acid encoding human hypoxia-inducible factor 2 alpha.

The PCT publication WO 01/62965 discloses and claims a differentialscreening method for identifying a genetic element involved in acellular process, which method includes introducing HIF2α into cells(Kingsman, 2001). This publication also discloses the development ofHIF2α agonists or antagonists.

The PCT publication WO 02/34291 claims methods and reagents, includingthe use of antisense oligonucleotides, for the inhibition of human HIF1αtranscription (Colgan, 2002). This publication also discloses a nucleicacid encoding human hypoxia-inducible factor 2 alpha.

U.S. Pat. No. 6,395,548 claims a nucleic acid encoding a deletion mutantof human HIF2α and the use of this deletion mutant as a method ofinhibiting expression of an angiogenic factor in vitro. This patent alsodiscloses a nucleic acid encoding human hypoxia-inducible factor 2alpha, as well as nucleic acids complementary to all or part of thehuman HIF2α cDNA for use in antisense treatment to inhibit theexpression of HIF2α (Lee et al., 2002).

U.S. Pat. No. 6,432,927 discloses nucleic acid sequences, includingsense and antisense oligonucleotides, which are derived from an HIF2αand incorporated into recombinant nucleic acid molecules for the purposeof sustaining HIF2α expression in cells (Gregory and Vincent, 2002).

The nucleic acid sequence encoding a human HIF2α and insertion of thissequence into a viral expression vector, for the purpose of drivinghuman HIF2α expression in mammalian cells, is disclosed in the PCTpublication WO 02/068466 (White et al., 2002).

The PCT publication WO 02/094862 discloses a method for introducing intoa muscle cell a nucleic acid sequence encoding hypoxia-inducible factor2 alpha, for the purpose of overexpressing HIF2α and stimulatingangiogenesis or metabolic activity (Guy, 2002).

Disclosed and claimed in the US pre-grant publication 2003/0045686 is anucleic acid encoding human hypoxia-inducible factor 2 alpha, and amethod of delivering a therapeutically effective amount of this nucleicacid to a subject for the purpose of reducing or preventing hypoxia(Kaelin Jr. and Ivan, 2003). This publication also discloses and claimshuman HIF muteins, including HIF2α mutein, which are designed to be morestable and/or resistant to degradation.

As a consequence of HIF2α involvement in many diseases, there remains along felt need for additional agents capable of effectively regulatingHIF2α function. As such, inhibition is especially important in thetreatment of cancer, given that the upregulation of expression of HIF2αis associated with so many different types of cancer.

As a consequence of HIF1α and HIF2α involvement in many diseases, thereremains a long felt need for additional agents capable of effectivelyinhibiting HIF1α and HIF2α function.

Antisense technology is emerging as an effective means for reducing theexpression of specific gene products and may therefore prove to beuniquely useful in a number of therapeutic, diagnostic, and researchapplications for the modulation of HIF1α and HIF2α expression.

The present invention provides compositions and methods for modulatingHIF1α and HIF2α expression. In particular antisense compositions formodulating HIF1α and/or HIF2α expression are believed to be useful intreatment of abnormal proliferative conditions associated with HIF1αand/or HIF2α. Examples of abnormal proliferative conditions arehyperproliferative disorders such as cancers, tumors, hyperplasias,pulmonary fibrosis, angiogenesis, psoriasis, atherosclerosis and smoothmuscle cell proliferation in the blood vessels, such as stenosis orrestenosis following angioplasty. It is presently believed thatinhibition of both HIF1α and HIF2α may be a particularly useful approachto treatment of such disorders.

SUMMARY OF THE INVENTION

The present invention is directed to compounds, especially nucleic acidand nucleic acid-like oligomers, which are targeted to a nucleic acidencoding HIF1α and/or HIF2α, and which modulate the expression of HIF1αand/or HIF2α. Pharmaceutical and other compositions comprising thecompounds of the invention are also provided. Further provided aremethods of screening for modulators of HIF1α and/or HIF2α and methods ofmodulating the expression of HIF1α and/or HIF2α in cells, tissues oranimals comprising contacting said cells, tissues or animals with one ormore of the compounds or compositions of the invention. Methods oftreating an animal, particularly a human, suspected of having or beingprone to a disease or condition associated with expression of HIF1αand/or HIF2α are also set forth herein. Such methods compriseadministering a therapeutically or prophylactically effective amount ofone or more of the compounds or compositions of the invention to theperson in need of treatment.

DETAILED DESCRIPTION OF THE INVENTION

A. Overview of the Invention

The present invention employs compounds, preferably oligonucleotides andsimilar species for use in modulating the function or effect of nucleicacid molecules encoding HIF1α or HIF2α. This is accomplished byproviding oligonucleotides which specifically hybridize with one or morenucleic acid molecules encoding HIF1α or HIF2α. Thus “target nucleicacid” refers to a nucleic acid molecule encoding HIF1α or HIF2α. As usedherein, the term “nucleic acid molecule encoding HIF1α” has been usedfor convenience to encompass DNA encoding HIF1α, RNA (including pre-mRNAand mRNA or portions thereof) transcribed from such DNA, and also cDNAderived from such RNA. Similarly, the term “nucleic acid moleculeencoding HIF2α” has been used for convenience to encompass DNA encodingHIF2α, RNA (including pre-mRNA and mRNA or portions thereof) transcribedfrom such DNA, and also cDNA derived from such RNA. The hybridization ofa compound of this invention with its target nucleic acid is generallyreferred to as “antisense”. Consequently, the preferred mechanismbelieved to be included in the practice of some preferred embodiments ofthe invention is referred to herein as “antisense inhibition.” Suchantisense inhibition is typically based upon hydrogen bonding-basedhybridization of oligonucleotide strands or segments such that at leastone strand or segment is cleaved, degraded, or otherwise renderedinoperable. In this regard, it is presently preferred to target specificnucleic acid molecules and their functions for such antisenseinhibition.

The functions of DNA to be interfered with can include replication andtranscription. Replication and transcription, for example, can be froman endogenous cellular template, a vector, a plasmid construct orotherwise. The functions of RNA to be interfered with can includefunctions such as translocation of the RNA to a site of proteintranslation, translocation of the RNA to sites within the cell which aredistant from the site of RNA synthesis, translation of protein from theRNA, splicing of the RNA to yield one or more RNA species, and catalyticactivity or complex formation involving the RNA which may be engaged inor facilitated by the RNA. One preferred result of such interferencewith target nucleic acid function is modulation of the expression ofHIF1α or HIF2α. In the context of the present invention, “modulation”and “modulation of expression” mean either an increase (stimulation) ora decrease (inhibition) in the amount or levels of a nucleic acidmolecule encoding the gene, e.g., DNA or RNA. Inhibition is often thepreferred form of modulation of expression and mRNA is often a preferredtarget nucleic acid.

In the context of this invention, “hybridization” means the pairing ofcomplementary strands of oligomeric compounds. In the present invention,the preferred mechanism of pairing involves hydrogen bonding, which maybe Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,between complementary nucleoside or nucleotide bases (nucleobases) ofthe strands of oligomeric compounds. For example, adenine and thymineare complementary nucleobases which pair through the formation ofhydrogen bonds. Hybridization can occur under varying circumstances.

An antisense compound is specifically hybridizable when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a loss of activity, and there is asufficient degree of complementarity to avoid non-specific binding ofthe antisense compound to non-target nucleic acid sequences underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

In the present invention the phrase “stringent hybridization conditions”or “stringent conditions” refers to conditions under which a compound ofthe invention will hybridize to its target sequence, but to a minimalnumber of other sequences. Stringent conditions are sequence-dependentand will be different in different circumstances and in the context ofthis invention, “stringent conditions” under which oligomeric compoundshybridize to a target sequence are determined by the nature andcomposition of the oligomeric compounds and the assays in which they arebeing investigated.

“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleobases of an oligomeric compound. For example,if a nucleobase at a certain position of an oligonucleotide (anoligomeric compound), is capable of hydrogen bonding with a nucleobaseat a certain position of a target nucleic acid, said target nucleic acidbeing a DNA, RNA, or oligonucleotide molecule, then the position ofhydrogen bonding between the oligonucleotide and the target nucleic acidis considered to be a complementary position. The oligonucleotide andthe further DNA, RNA, or oligonucleotide molecule are complementary toeach other when a sufficient number of complementary positions in eachmolecule are occupied by nucleobases which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of precise pairing orcomplementarity over a sufficient number of nucleobases such that stableand specific binding occurs between the oligonucleotide and a targetnucleic acid.

It is understood in the art that the sequence of an antisense compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligonucleotide may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure orhairpin structure). It is preferred that the antisense compounds of thepresent invention comprise at least 70% sequence complementarity to atarget region within the target nucleic acid, more preferably that theycomprise 90% sequence complementarity and even more preferably comprise95% sequence complementarity to the target region within the targetnucleic acid sequence to which they are targeted. For example, anantisense compound in which 18 of 20 nucleobases of the antisensecompound are complementary to a target region, and would thereforespecifically hybridize, would represent 90 percent complementarity. Inthis example, the remaining noncomplementary nucleobases may beclustered or interspersed with complementary nucleobases and need not becontiguous to each other or to complementary nucleobases. As such, anantisense compound which is 18 nucleobases in length having 4 (four)noncomplementary nucleobases which are flanked by two regions ofcomplete complementarity with the target nucleic acid would have 77.8%overall complementarity with the target nucleic acid and would thus fallwithin the scope of the present invention. Percent complementarity of anantisense compound with a region of a target nucleic acid can bedetermined routinely using BLAST programs (basic local alignment searchtools) and PowerBLAST programs known in the art (Altschul et al., J.Mol. Biol., 1990, 215, 403–410; Zhang and Madden, Genome Res., 1997, 7,649–656).

B. Compounds of the Invention

According to the present invention, compounds include antisenseoligomeric compounds, antisense oligonucleotides, ribozymes, externalguide sequence (EGS) oligonucleotides, alternate splicers, primers,probes, and other oligomeric compounds which hybridize to at least aportion of the target nucleic acid. As such, these compounds may beintroduced in the form of single-stranded, double-stranded, circular orhairpin oligomeric compounds and may contain structural elements such asinternal or terminal bulges or loops. Once introduced to a system, thecompounds of the invention may elicit the action of one or more enzymesor structural proteins to effect modification of the target nucleicacid.

One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded antisense compounds which are“DNA-like” elicit RNAse H. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. Similar roleshave been postulated for other ribonucleases such as those in the RNaseIII and ribonuclease L family of enzymes.

While the preferred form of antisense compound is a single-strandedantisense oligonucleotide, in many species the introduction ofdouble-stranded structures, such as double-stranded RNA (dsRNA)molecules, has been shown to induce potent and specificantisense-mediated reduction of the function of a gene or its associatedgene products. This phenomenon occurs in both plants and animals and isbelieved to have an evolutionary connection to viral defense andtransposon silencing.

The first evidence that dsRNA could lead to gene silencing in animalscame in 1995 from work in the nematode, Caenorhabditis elegans (Guo andKempheus, Cell, 1995; 81, 611–620). Montgomery et al. have shown thatthe primary interference effects of dsRNA are posttranscriptional(Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502–15507).The posttranscriptional antisense mechanism defined in Caenorhabditiselegans resulting from exposure to double-stranded RNA (dsRNA) has sincebeen designated RNA interference (RNAi). This term has been generalizedto mean antisense-mediated gene silencing involving the introduction ofdsRNA leading to the sequence-specific reduction of endogenous targetedmRNA levels (Fire et al., Nature, 1998, 391, 806–811). Recently, it hasbeen shown that it is, in fact, the single-stranded RNA oligomers ofantisense polarity of the dsRNAs which are the potent inducers of RNAi(Tijsterman et al., Science, 2002, 295, 694–697).

The oligonucleotides of the present invention also include variants inwhich a different base is present at one or more of the nucleotidepositions in the oligonucleotide. For example, if the first nucleotideis an adenosine, variants may be produced which contain thymidine,guanosine or cytidine at this position. This may be done at any of thepositions of the oligonucleotide. These oligonucleotides are then testedusing the methods described herein to determine their ability to inhibitexpression of HIF2α mRNA.

In the context of this invention, the term “oligomeric compound” refersto a polymer or oligomer comprising a plurality of monomeric units. Inthe context of this invention, the term “oligonucleotide” refers to anoligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid(DNA) or mimetics, chimeras, analogs and homologs thereof. This termincludes oligonucleotides composed of naturally occurring nucleobases,sugars and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for a targetnucleic acid and increased stability in the presence of nucleases.

While oligonucleotides are a preferred form of the compounds of thisinvention, the present invention comprehends other families of compoundsas well, including but not limited to oligonucleotide analogs andmimetics such as those described herein.

The compounds in accordance with this invention preferably comprise fromabout 8 to about 80 nucleobases (i.e. from about 8 to about 80 linkednucleosides). One of ordinary skill in the art will appreciate that theinvention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.

In one preferred embodiment, the compounds of the invention are 12 to 50nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases inlength.

In another preferred embodiment, the compounds of the invention are 15to 30 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

Particularly preferred compounds are oligonucleotides from about 12 toabout 50 nucleobases, even more preferably those comprising from about15 to about 30 nucleobases.

Antisense compounds 8–80 nucleobases in length comprising a stretch ofat least eight (8) consecutive nucleobases selected from within theillustrative antisense compounds are considered to be suitable antisensecompounds as well.

Exemplary preferred antisense compounds include oligonucleotidesequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred antisense compounds(the remaining nucleobases being a consecutive stretch of the sameoligonucleotide beginning immediately upstream of the 5′-terminus of theantisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the oligonucleotide contains about 8to about 80 nucleobases). Similarly preferred antisense compounds arerepresented by oligonucleotide sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of one of the illustrativepreferred antisense compounds (the remaining nucleobases being aconsecutive stretch of the same oligonucleotide beginning immediatelydownstream of the 3′-terminus of the antisense compound which isspecifically hybridizable to the target nucleic acid and continuinguntil the oligonucleotide contains about 8 to about 80 nucleobases). Onehaving skill in the art armed with the preferred antisense compoundsillustrated herein will be able, without undue experimentation, toidentify further preferred antisense compounds.

C. Targets of the Invention

“Targeting” an antisense compound to a particular nucleic acid molecule,in the context of this invention, can be a multistep process. Theprocess usually begins with the identification of a target nucleic acidwhose function is to be modulated. This target nucleic acid may be, forexample, a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state, ora nucleic acid molecule from an infectious agent. In the presentinvention, the target nucleic acid encodes HIF1α or HIF2α.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. Within the context of the presentinvention, the term “region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. Within regions of target nucleic acids are segments.“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid. “Sites,” as used in the present invention, aredefined as positions within a target nucleic acid.

Since, as is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes). It isalso known in the art that eukaryotic and prokaryotic genes may have twoor more alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of theinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAtranscribed from a gene encoding HIF1α or HIF2α, regardless of thesequence(s) of such codons. It is also known in the art that atranslation termination codon (or “stop codon”) of a gene may have oneof three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions which may betargeted effectively with the antisense compounds of the presentinvention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, apreferred region is the intragenic region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of agene.

Other target regions include the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene), and the 3′ untranslated region(3′UTR), known in the art to refer to the portion of an mRNA in the 3′direction from the translation termination codon, and thus includingnucleotides between the translation termination codon and 3′ end of anmRNA (or corresponding nucleotides on the gene). The 5′ cap site of anmRNA comprises an N7-methylated guanosine residue joined to the 5′-mostresidue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap regionof an mRNA is considered to include the 5′ cap structure itself as wellas the first 50 nucleotides adjacent to the cap site. It is alsopreferred to target the 5′ cap region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. Targeting splice sites, i.e.,intron-exon junctions or exon-intron junctions, may also be particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular splice product is implicatedin disease. Aberrant fusion junctions due to rearrangements or deletionsare also preferred target sites. mRNA transcripts produced via theprocess of splicing of two (or more) mRNAs from different gene sourcesare known as “fusion transcripts”. It is also known that introns can beeffectively targeted using antisense compounds targeted to, for example,DNA or pre-mRNA.

It is also known in the art that alternative RNA transcripts can beproduced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequence.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

It is also known in the art that variants can be produced through theuse of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites. Within thecontext of the invention, the types of variants described herein arealso preferred target nucleic acids.

The locations on the target nucleic acid to which the preferredantisense compounds hybridize are hereinbelow referred to as “preferredtarget segments.” As used herein the term “preferred target segment” isdefined as at least an 8-nucleobase portion of a target region to whichan active antisense compound is targeted. While not wishing to be boundby theory, it is presently believed that these target segments representportions of the target nucleic acid which are accessible forhybridization.

While the specific sequences of certain preferred target segments areset forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional preferred target segments may beidentified by one having ordinary skill.

Target segments 8–80 nucleobases in length comprising a stretch of atleast eight (8) consecutive nucleobases selected from within theillustrative preferred target segments are considered to be suitable fortargeting as well.

Target segments can include DNA or RNA sequences that comprise at leastthe 8 consecutive nucleobases from the 5′-terminus of one of theillustrative preferred target segments (the remaining nucleobases beinga consecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 8 to about 80 nucleobases). Similarlypreferred target segments are represented by DNA or RNA sequences thatcomprise at least the 8 consecutive nucleobases from the 3′-terminus ofone of the illustrative preferred target segments (the remainingnucleobases being a consecutive stretch of the same DNA or RNA beginningimmediately downstream of the 3′-terminus of the target segment andcontinuing until the DNA or RNA contains about 8 to about 80nucleobases). One having skill in the art armed with the preferredtarget segments illustrated herein will be able, without undueexperimentation, to identify further preferred target segments.

Once one or more target regions, segments or sites have been identified,antisense compounds are chosen which are sufficiently complementary tothe target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

D. Screening and Target Validation

In a further embodiment, the “preferred target segments” identifiedherein may be employed in a screen for additional compounds thatmodulate the expression of HIF1α or HIF2α. “Modulators” are thosecompounds that decrease or increase the expression of a nucleic acidmolecule encoding HIF1α or HIF2α and which comprise at least an8-nucleobase portion which is complementary to a preferred targetsegment. The screening method comprises the steps of contacting apreferred target segment of a nucleic acid molecule encoding HIF1α orHIF2α with one or more candidate modulators, and selecting for one ormore candidate modulators which decrease or increase the expression of anucleic acid molecule encoding HIF1α or HIF2α. Once it is shown that thecandidate modulator or modulators are capable of modulating (e.g. eitherdecreasing or increasing) the expression of a nucleic acid moleculeencoding HIF1α or HIF2α, the modulator may then be employed in furtherinvestigative studies of the function of HIF1α or HIF2α, or for use as aresearch, diagnostic, or therapeutic agent in accordance with thepresent invention.

The preferred target segments of the present invention may be also becombined with their respective complementary antisense compounds of thepresent invention to form stabilized double-stranded (duplexed)oligonucleotides.

Such double stranded oligonucleotide moieties have been shown in the artto modulate target expression and regulate translation as well as RNAprocessing via an antisense mechanism. Moreover, the double-strandedmoieties may be subject to chemical modifications (Fire et al., Nature,1998, 391, 806–811; Timmons and Fire, Nature 1998, 395, 854; Timmons etal., Gene, 2001, 263, 103–112; Tabara et al., Science, 1998, 282,430–431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95,15502–15507; Tuschl et al., Genes Dev., 1999, 13, 3191–3197; Elbashir etal., Nature, 2001, 411, 494–498; Elbashir et al., Genes Dev. 2001, 15,188–200). For example, such double-stranded moieties have been shown toinhibit the target by the classical hybridization of antisense strand ofthe duplex to the target, thereby triggering enzymatic degradation ofthe target (Tijsterman et al., Science, 2002, 295, 694–697).

The compounds of the present invention can also be applied in the areasof drug discovery and target validation. The present inventioncomprehends the use of the compounds and preferred target segmentsidentified herein in drug discovery efforts to elucidate relationshipsthat exist between HIF1α or HIF2α and a disease state, phenotype, orcondition. These methods include detecting or modulating HIF1α or HIF2αcomprising contacting a sample, tissue, cell, or organism with thecompounds of the present invention, measuring the nucleic acid orprotein level of HIF1α or HIF2α and/or a related phenotypic or chemicalendpoint at some time after treatment, and optionally comparing themeasured value to a non-treated sample or sample treated with a furthercompound of the invention. These methods can also be performed inparallel or in combination with other experiments to determine thefunction of unknown genes for the process of target validation or todetermine the validity of a particular gene product as a target fortreatment or prevention of a particular disease, condition, orphenotype.

E. Kits, Research Reagents, Diagnostics, and Therapeutics

The compounds of the present invention can be utilized for diagnostics,therapeutics, prophylaxis and as research reagents and kits.Furthermore, antisense oligonucleotides, which are able to inhibit geneexpression with exquisite specificity, are often used by those ofordinary skill to elucidate the function of particular genes or todistinguish between functions of various members of a biologicalpathway.

For use in kits and diagnostics, the compounds of the present invention,either alone or in combination with other compounds or therapeutics, canbe used as tools in differential and/or combinatorial analyses toelucidate expression patterns of a portion or the entire complement ofgenes expressed within cells and tissues.

As one nonlimiting example, expression patterns within cells or tissuestreated with one or more antisense compounds are compared to controlcells or tissues not treated with antisense compounds and the patternsproduced are analyzed for differential levels of gene expression as theypertain, for example, to disease association, signaling pathway,cellular localization, expression level, size, structure or function ofthe genes examined. These analyses can be performed on stimulated orunstimulated cells and in the presence or absence of other compoundswhich affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17–24; Celis, et al., FEBS Lett., 2000, 480, 2–16), SAGE (serialanalysis of gene expression) (Madden, et al., Drug Discov. Today, 2000,5, 415–425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258–72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976–81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2–16; Jungblut, et al., Electrophoresis,1999, 20, 2100–10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2–16; Larsson, et al., J. Biotechnol., 2000,80, 143–57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91–98; Larson, et al., Cytometry, 2000, 41,203–208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316–21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286–96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895–904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235–41).

The compounds of the invention are useful for research and diagnostics,because these compounds hybridize to nucleic acids encoding HIF1α orHIF2α. For example, oligonucleotides that are shown to hybridize withsuch efficiency and under such conditions as disclosed herein as to beeffective HIF1α or HIF2α inhibitors will also be effective primers orprobes under conditions favoring gene amplification or detection,respectively. These primers and probes are useful in methods requiringthe specific detection of nucleic acid molecules encoding HIF1α or HIF2αand in the amplification of said nucleic acid molecules for detection orfor use in further studies of HIF1α or HIF2α. Hybridization of theantisense oligonucleotides, particularly the primers and probes, of theinvention with a nucleic acid encoding HIF1α or HIF2α can be detected bymeans known in the art. Such means may include conjugation of an enzymeto the oligonucleotide, radiolabelling of the oligonucleotide or anyother suitable detection means. Kits using such detection means fordetecting the level of HIF1α or HIF2α in a sample may also be prepared.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense compounds have beenemployed as therapeutic moieties in the treatment of disease states inanimals, including humans. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat antisense compounds can be useful therapeutic modalities that canbe configured to be useful in treatment regimes for the treatment ofcells, tissues and animals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having adisease or disorder which can be treated by modulating the expression ofHIF1α or HIF2α is treated by administering one or more antisensecompounds in accordance with this invention. For example, in onenon-limiting embodiment, the methods comprise the step of administeringto the animal in need of treatment, a therapeutically effective amountof a HIF1α or HIF2α inhibitor. The HIF1α or HIF2α inhibitors of thepresent invention effectively inhibit the activity of the HIF targetprotein or inhibit the expression of the HIF1α or HIF2α protein. In oneembodiment, the activity or expression of HIF1α or HIF2α in an animal isinhibited by about 10%. Preferably, the activity or expression of HIF1αor HIF2α in an animal is inhibited by about 30%. More preferably, theactivity or expression of HIF1α and/or HIF2α in an animal is inhibitedby 50% or more.

For example, the reduction of the expression of HIF1α may be measured inserum, adipose tissue, liver or any other body fluid, tissue or organ ofthe animal. Preferably, the cells contained within said fluids, tissuesor organs being analyzed contain a nucleic acid molecule encoding HIF1αor HIF2α protein and/or the HIF1α or HIF2α protein itself.

The compounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of a compound to a suitablepharmaceutically acceptable diluent or carrier. Use of the compounds andmethods of the invention may also be useful prophylactically.

F. Modifications

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound,however, linear compounds are generally preferred. In addition, linearcompounds may have internal nucleobase complementarity and may thereforefold in a manner as to produce a fully or partially double-strandedcompound. Within oligonucleotides, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

Modified Internucleoside Linkages (Backbones)

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones containing a phosphorusatom therein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

Modified Sugar and Internucleoside Linkages-Mimetics

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage (i.e. the backbone), of the nucleotide units arereplaced with novel groups. The nucleobase units are maintained forhybridization with an appropriate target nucleic acid. One suchcompound, an oligonucleotide mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotideis replaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497–1500.

Preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified Sugars

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Otherpreferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486–504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have. sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

A further preferred modification of the sugar includes Locked NucleicAcids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety.The linkage is preferably a methylene (—CH₂—)_(n) group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

Natural and Modified Nucleobases

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further modified nucleobases include tricyclicpyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858–859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289–302, Crooke, S. T. and Lebleu, B.ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6–1.2° C.and are presently preferred base substitutions, even more particularlywhen combined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

Conjugates

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. These moieties or conjugates can includeconjugate groups covalently bound to functional groups such as primaryor secondary hydroxyl groups. Conjugate groups of the invention includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of thisinvention, include groups that improve uptake, enhance resistance todegradation, and/or strengthen sequence-specific hybridization with thetarget nucleic acid. Groups that enhance the pharmacokinetic properties,in the context of this invention, include groups that improve uptake,distribution, metabolism or excretion of the compounds of the presentinvention. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992,and U.S. Pat. No. 6,287,860, the entire disclosure of which areincorporated herein by reference. Conjugate moieties include but are notlimited to lipid moieties such as a cholesterol moiety, cholic acid, athioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphaticchain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Oligonucleotides of the invention may also be conjugated to active drugsubstances, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic. Oligonucleotide-drug conjugates andtheir preparation are described in U.S. patent application Ser. No.09/334,130 (filed Jun. 15, 1999) which is incorporated herein byreference in its entirety.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

Chimeric Compounds

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide.

The present invention also includes antisense compounds which arechimeric compounds. “Chimeric” antisense compounds or “chimeras,” in thecontext of this invention, are antisense compounds, particularlyoligonucleotides, which contain two or more chemically distinct regions,each made up of at least one monomer unit, i.e., a nucleotide in thecase of an oligonucleotide compound. These oligonucleotides typicallycontain at least one region wherein the oligonucleotide is modified soas to confer upon the oligonucleotide increased resistance to nucleasedegradation, increased cellular uptake, increased stability and/orincreased binding affinity for the target nucleic acid. An additionalregion of the oligonucleotide may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as RNAseL which cleaves both cellularand viral RNA. Cleavage of the RNA target can be routinely detected bygel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

G. Formulations

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof. Accordingly, for example, the disclosure is alsodrawn to prodrugs and pharmaceutically acceptable salts of the compoundsof the invention, pharmaceutically acceptable salts of such prodrugs,and other bioequivalents. The term “prodrug” indicates a therapeuticagent that is prepared in an inactive form that is converted to anactive form (i.e., drug) within the body or cells thereof by the actionof endogenous enzymes or other chemicals and/or conditions. Inparticular, prodrug versions of the oligonucleotides of the inventionare prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivativesaccording to the methods disclosed in WO 93/24510 to Gosselin et al.,published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 toImbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Foroligonucleotides, preferred examples of pharmaceutically acceptablesalts and their uses are further described in U.S. Pat. No. 6,287,860,which is incorporated herein in its entirety.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration, includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2′-O-methoxyethyl modification are believed to beparticularly useful for oral administration. Pharmaceutical compositionsand formulations for topical administration may include transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Coated condoms, gloves and the like may also be useful.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogenous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug which may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes which are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome comprises oneor more glycolipids or is derivatized with one or more hydrophilicpolymers, such as a polyethylene glycol (PEG) moiety. Liposomes andtheir uses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating non-surfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

Preferred formulations for topical administration include those in whichthe oligonucleotides of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Preferred lipids andliposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA).

For topical or other administration, oligonucleotides of the inventionmay be encapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters, pharmaceutically acceptable salts thereof, and theiruses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety. Topical formulations are describedin detail in U.S. patent application Ser. No. 09/315,298 filed on May20, 1999, which is incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Preferred bileacids/salts and fatty acids and their uses are further described in U.S.Pat. No. 6,287,860, which is incorporated herein in its entirety. Alsopreferred are combinations of penetration enhancers, for example, fattyacids/salts in combination with bile acids/salts. A particularlypreferred combination is the sodium salt of lauric acid, capric acid andUDCA. Further penetration enhancers include polyoxyethylene-9-laurylether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the inventionmay be delivered orally, in granular form including sprayed driedparticles, or complexed to form micro or nanoparticles. Oligonucleotidecomplexing agents and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety. Oralformulations for oligonucleotides and their preparation are described indetail in U.S. application Ser. No. 09/108,673 (filed Jul. 1, 1998),Ser. No. 09/315,298 (filed May 20, 1999) and Ser. No. 10/071,822, filedFeb. 8, 2002, each of which is incorporated herein by reference in theirentirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more oligomeric compounds and one or more otherchemotherapeutic agents which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited tocancer chemotherapeutic drugs such as daunorubicin, daunomycin,dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,bleomycin, mafosfamide, ifosfamide, cytosine ara-binoside,bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxyco-formycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teni-poside, cisplatin and diethylstilbestrol(DES). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of antisense compounds and other non-antisense drugs arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Alternatively, compositions ofthe invention may contain two or more antisense compounds targeted todifferent regions of the same nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

H. Dosing

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC₅₀s found to be effective inin vitro and in vivo animal models. In general, dosage is from 0.01 ugto 100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. Persons ofordinary skill in the art can easily estimate repetition rates fordosing based on measured residence times and concentrations of the drugin bodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Synthesis of Nucleoside Phosphoramidites

The following compounds, including amidites and their intermediates wereprepared as described in U.S. Pat. No. 6,426,220 and published PCT WO02/36743; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dCamidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for5-methyl-dC amidite,5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine,2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modifiedamidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate,5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite),5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidineintermediate,5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidinepenultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amdite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites,2′-(Dimethylaminooxyethoxy) nucleoside amidites,5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine ,5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine,2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine,5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine,2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-(Aminooxyethoxy) nucleoside amidites,N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites,2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine,5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine and5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2 Oligonucleotide and Oligonucleoside Synthesis

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

Oligonucleotides:

Unsubstituted and substituted phosphodiester (P═O) oligonucleotides aresynthesized on an automated DNA synthesizer (Applied Biosystems model394) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12–16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Oligonucleosides:

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 3 RNA Synthesis

In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

RNA oligonucleotides are synthesized in a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5′-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3′-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5′-end of the first nucleoside. Thesupport is washed and any unreacted 5′-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5′-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

Following synthesis, the methyl protecting groups on the phosphates arecleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

The 2′-orthoester groups are the last protecting groups to be removed.The ethylene glycol monoacetate orthoester protecting group developed byDharmacon Research, Inc. (Lafayette, Colo.), is one example of a usefulorthoester protecting group which, has the following importantproperties. It is stable to the conditions of nucleoside phosphoramiditesynthesis and oligonucleotide synthesis. However, after oligonucleotidesynthesis the oligonucleotide is treated with methylamine which not onlycleaves the oligonucleotide from the solid support but also removes theacetyl groups from the orthoesters. The resulting 2-ethyl-hydroxylsubstituents on the orthoester are less electron withdrawing than theacetylated precursor. As a result, the modified orthoester becomes morelabile to acid-catalyzed hydrolysis. Specifically, the rate of cleavageis approximately 10 times faster after the acetyl groups are removed.Therefore, this orthoester possesses sufficient stability in order to becompatible with oligonucleotide synthesis and yet, when subsequentlymodified, permits deprotection to be carried out under relatively mildaqueous conditions compatible with the final RNA oligonucleotideproduct.

Additionally, methods of RNA synthesis are well known in the art(Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe,S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820–11821; Matteucci, M.D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185–3191;Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22,1859–1862; Dahl, B. J., et al., Acta Chem. Scand., 1990, 44, 639–641;Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311–4314; Wincott,F. et al., Nucleic Acids Res., 1995, 23, 2677–2684; Griffin, B. E., etal., Tetrahedron, 1967, 23, 2301–2313; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2315–2331).

RNA antisense compounds (RNA oligonucleotides) of the present inventioncan be synthesized by the methods herein or purchased from DharmaconResearch, Inc (Lafayette, Colo.). Once synthesized, complementary RNAantisense compounds can then be annealed by methods known in the art toform double stranded (duplexed) antisense compounds. For example,duplexes can be formed by combining 30 μl of each of the complementarystrands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and15 μl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOHpH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90°C., then 1 hour at 37° C. The resulting duplexed antisense compounds canbe used in kits, assays, screens, or other methods to investigate therole of a target nucleic acid.

Example 4 Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12–16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)] chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5 D Sign and Screening of Duplexed Antisense Compounds TargetingHIF1α or HIF2α

In accordance with the present invention, a series of nucleic acidduplexes comprising the antisense compounds of the present invention andtheir complements can be designed to target HIF1α or HIF2α. Thenucleobase sequence of the antisense strand of the duplex preferablycomprises at least a portion of an oligonucleotide in Tables 1, 3, 4, 5,6, 13, or 14. The ends of the strands may be modified by the addition ofone or more natural or modified nucleobases to form an overhang. Thesense strand of the dsRNA is then designed and synthesized as thecomplement of the antisense strand and may also contain modifications oradditions to either terminus. For example, in one embodiment, bothstrands of the dsRNA duplex would be complementary over the centralnucleobases, each having overhangs at one or both termini.

For example, a duplex comprising an antisense strand having the sequenceCGAGAGGCGGACGGGACCG (SEQ ID NO: 455) and having a two-nucleobaseoverhang of deoxythymidine(dT) would have the following structure:

As another example, a duplex comprising an antisense strand having thesequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 455) and having no overhangswould have the following structure:

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from Dharmacon Research Inc., (Lafayette, Colo.). Oncesynthesized, the complementary strands are annealed. The single strandsare aliquotted and diluted to a concentration of 50 uM. Once diluted, 30uL of each strand is combined with 15uL of a 5× solution of annealingbuffer. The final concentration of said buffer is 100 mM potassiumacetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The finalvolume is 75 uL. This solution is incubated for 1 minute at 90° C. andthen centrifuged for 15 seconds. The tube is allowed to sit for 1 hourat 37° C. at which time the dsRNA duplexes are used in experimentation.The final concentration of the dsRNA duplex is 20 uM. This solution canbe stored frozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the duplexed antisense compounds are evaluated for theirability to modulate HIF1● or HIF2● expression.

When cells reached 80% confluency, they are treated with duplexedantisense compounds of the invention. For cells grown in 96-well plates,wells are washed once with 200 ●L OPTI-MEM-1 reduced-serum medium (GibcoBRL) and then treated with 130 ●L of OPTI-MEM-1 containing 12 ●g/mLLIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at afinal concentration of 200 M. After 5 hours of treatment, the medium isreplaced with fresh medium. Cells are harvested 16 hours aftertreatment, at which time RNA is isolated and target reduction measuredby RT-PCR.

Example 6 Oligonucleotide Isolation

After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12–16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis was determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32 +/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162–18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a 96-well format. Phosphodiester internucleotidelinkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55–60° C.) for 12–16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis—96-Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, ribonucleaseprotection assays, or RT-PCR.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #353872) at a density of7000 cells/well for use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 Cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville, Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville, Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

b.END Cells:

The mouse brain endothelial cell line b.END was obtained from Dr. WernerRisau at the Max Plank Institute (Bad Nauheim, Germany). b.END cellswere routinely cultured in DMEM, high glucose (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 3000cells/well for use in RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

Treatment with Antisense Compounds:

When cells reached 65–75% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation,Carlsbad, Calif.) and the desired concentration of oligonucleotide.Cells are treated and data are obtained in triplicate. After 4–7 hoursof treatment at 37° C., the medium was replaced with fresh medium. Cellswere harvested 16–24 hours after oligonucleotide treatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) orc-raf (for ISIS 15770) mRNA is then utilized as the screeningconcentration for new oligonucleotides in subsequent experiments forthat cell line. If 80% inhibition is not achieved, the lowestconcentration of positive control oligonucleotide that results in 60%inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as theoligonucleotide screening concentration in subsequent experiments forthat cell line. If 60% inhibition is not achieved, that particular cellline is deemed as unsuitable for oligonucleotide transfectionexperiments. The concentrations of antisense oligonucleotides usedherein are from 50 nM to 300 nM.

Example 10 Analysis of Oligonucleotide Inhibition of HIF1α and/or HIF2αExpression

Antisense modulation of HIF1α and/or HIF2α expression can be assayed ina variety of ways known in the art. For example, HIF1α or HIF2α mRNAlevels can be quantitated by, e.g., Northern blot analysis, competitivepolymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-timequantitative PCR is presently preferred. RNA analysis can be performedon total cellular RNA or poly(A)+ mRNA. The preferred method of RNAanalysis of the present invention is the use of total cellular RNA asdescribed in other examples herein. Methods of RNA isolation are wellknown in the art. Northern blot analysis is also routine in the art.Real-time quantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7600, 7700, or 7900 Sequence DetectionSystem, available from PE-Applied Biosystems, Foster City, Calif. andused according to manufacturer's instructions.

Protein levels of HIF1α or HIF2α can be quantitated in a variety of wayswell known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS). Antibodies directed to HIF1αor HIF2α can be identified and obtained from a variety of sources, suchas the MSRS catalog of antibodies (Aerie Corporation, Birmingham,Mich.), or can be prepared via conventional monoclonal or polyclonalantibody generation methods well known in the art.

Example 11 Design of Phenotypic Assays and In Vivo Studies for the Useof HIF1α or HIF2α Inhibitors

Phenotypic Assays

Once HIF1α or HIF2α inhibitors have been identified by the methodsdisclosed herein, the compounds are further investigated in one or morephenotypic assays, each having measurable endpoints predictive ofefficacy in the treatment of a particular disease state or condition.

Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of HIF1α and/or HIF2α in health and disease.Representative phenotypic assays, which can be purchased from any one ofseveral commercial vendors, include those for determining cellviability, cytotoxicity, proliferation or cell survival (MolecularProbes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assaysincluding enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences,Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.),cell regulation, signal transduction, inflammation, oxidative processesand apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated with HIF1αand/or HIF2α inhibitors identified from the in vitro studies as well ascontrol compounds at optimal concentrations which are determined by themethods described above. At the end of the treatment period, treated anduntreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Analysis of the genotype of the cell (measurement of the expression ofone or more of the genes of the cell) after treatment is also used as anindicator of the efficacy or potency of the HIF1α and/or HIF2αinhibitors. Hallmark genes, or those genes suspected to be associatedwith a specific disease state, condition, or phenotype, are measured inboth treated and untreated cells.

Example 12 RNA Isolation

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem.,1996, 42, 1758–1764). Other methods for poly(A)+ mRNA isolation areroutine in the art. Briefly, for cells grown on 96-well plates, growthmedium was removed from the cells and each well was washed with 200 μLcold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 MNaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added toeach well, the plate was gently agitated and then incubated at roomtemperature for five minutes. 55 μL of lysate was transferred to Oligod(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates wereincubated for 60 minutes at room temperature, washed 3 times with 200 μLof wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After thefinal wash, the plate was blotted on paper towels to remove excess washbuffer and then air-dried for 5 minutes, 60 μL of elution buffer (5 mMTris-HCl pH 7.6), preheated to 70° C., was added to each well, the platewas incubated on a 90° C. hot plate for 5 minutes, and the eluate wasthen transferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia, Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 150 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 150 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 1 minute. 500 μL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and incubatedfor 15 minutes and the vacuum was again applied for 1 minute. Anadditional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE wasthen added to each well of the RNEASY 96™ plate and the vacuum appliedfor a period of 90 seconds. The Buffer RPE wash was then repeated andthe vacuum was applied for an additional 3 minutes. The plate was thenremoved from the QIAVAC™ manifold and blotted dry on paper towels. Theplate was then re-attached to the QIAVAC™ manifold fitted with acollection tube rack containing 1.2 mL collection tubes. RNA was theneluted by pipetting 140 μL of RNAse free water into each well,incubating 1 minute, and then applying the vacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13 Real-Time Quantitative PCR Analysis of HIF1α mRNA Levels

Quantitation of HIF1α mRNA levels was accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., FAM or JOE, obtained from either PE-AppliedBiosystems, Foster City, Calif., Operon Technologies Inc., Alameda,Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 3′ end of the probe. When the probeand dyes are intact, reporter dye emission is quenched by the proximityof the 3′ quencher dye. During amplification, annealing of the probe tothe target sequence creates a substrate that can be cleaved by the5′-exonuclease activity of Taq polymerase. During the extension phase ofthe PCR amplification cycle, cleavage of the probe by Taq polymerasereleases the reporter dye from the remainder of the probe (and hencefrom the quencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™Sequence Detection System. In each assay, a series of parallel reactionscontaining serial dilutions of mRNA from untreated control samplesgenerates a standard curve that is used to quantitate the percentinhibition after antisense oligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

PCR reagents were obtained from Invitrogen Corporation, (Carlsbad,Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail(2.5× PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of dATP, dCTP,dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nMof probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 UnitsMuLV reverse transcriptase, and 2.5× ROX dye) to 96-well platescontaining 30 μL total RNA solution (20–200 ng). The RT reaction wascarried out by incubation for 30 minutes at 48° C. Following a 10 minuteincubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of atwo-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by real time RT-PCR are normalized usingeither the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real timeRT-PCR, by being run simultaneously with the target, multiplexing, orseparately. Total RNA is quantified using RiboGreen™ RNA quantificationreagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNAquantification by RiboGreen™ are taught in Jones, L. J., et al,(Analytical Biochemistry, 1998, 265, 368–374).

In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nmand emission at 530 nm.

Probes and primers to human HIF1α were designed to hybridize to a humanHIF1α sequence, using published sequence information (GenBank accessionnumber U29165.1, incorporated herein by reference and incorporatedherein as SEQ ID NO:4). For human HIF1α the PCR primers were:

forward primer: CCAGTTACGTTCCTTCGATCAGT (SEQ ID NO: 5) reverse primer:TTTGAGGACTTGCGCTTTCA (SEQ ID NO: 6) and the PCR probe was:FAM-TCACCATTAGAAAGCAGTTCCGCAAGCC-TAMRA (SEQ ID NO: 7) where FAM is thefluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCRprimers were:forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8) reverse primer:GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the PCR probe was: 5′JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10) where JOE is thefluorescent reporter dye and TAMRA is the quencher dye.

Example 14 Northern Blot Analysis of HIF1α mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

To detect human HIF1α, a human HIF1α specific probe was prepared by PCRusing the forward primer CCAGTTACGTTCCTTCGATCAGT (SEQ ID NO: 5) and thereverse primer TTTGAGGACTTGCGCTTTCA (SEQ ID NO: 6). To normalize forvariations in loading and transfer efficiency membranes were strippedand probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH)RNA (Clontech, Palo Alto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15 Antisense Inhibition of Human HIF1α Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

In accordance with the present invention, a series of antisensecompounds were designed to target different regions of the human HIF1αRNA, using published sequences (GenBank accession number U29165.1,incorporated herein by reference and incorporated herein as SEQ ID NO:4, positions 82000 to 139500 of the sequence with GenBank accessionnumber AL137129.4, incorporated herein by reference and incorporatedherein as SEQ ID NO: 11, GenBank accession number AU123241.1,incorporated herein by reference and incorporated herein as SEQ ID NO:12, and GenBank accession number AB073325.1, incorporated herein byreference and incorporated herein as SEQ ID NO: 13). The compounds areshown in Table 1. “Target site” indicates the first (5′-most) nucleotidenumber on the particular target sequence to which the compound binds.All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on human HIF1α mRNA levels by quantitative real-time PCR asdescribed in other examples herein. Data are averages from threeexperiments in which A549 cells were treated with the antisenseoligonucleotides of the present invention. If present, “N.D.” indicates“no data”.

TABLE 1 Inhibition of human HIF1α mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET SEQ ID TARGET % SEQ ID ISIS # REGION NO SITE SEQUENCE INHIB NO175477 Coding 4 2496 aaagtgatgtagtagctgca 54 14 175478 Coding 4 854ggtatcatatacgtgaatgt 73 15 175479 3′UTR 4 3179 taccacgtactgctggcaaa 3116 175480 Coding 4 2039 tgtgctttgaggacttgcgc 94 17 175481 Coding 4 583gaaatgtaaatcatgtcacc 56 18 175482 Coding 4 1408 tcaaagaggctacttgtatc 7519 175483 Coding 4 1674 ttaatgcaacttcttgattg 45 20 175484 3′UTR 4 3333atcattattatatgattaac 60 21 175485 5′UTR 4 152 gaaaggcaagtccagaggtg 42 22175486 3′UTR 4 3027 taaactccctagccaaaaat 40 23 175487 Coding 4 2085cattagcagtaggttcttgt 75 24 175488 3′UTR 4 3101 gatcatgatgaaaggttact 8625 175489 Coding 4 1001 aaatttcatatccaggctgt 85 26 175490 Coding 4 460agtttcctcacacgcaaata 38 27 175491 Coding 4 1983 actgatcgaaggaacgtaac 8728 175492 Coding 4 2404 cgctttctctgagcattctg 44 29 175493 Coding 4 649aaatcaaacacactgtgtcc 79 30 175494 Coding 4 1139 tcctttagtaaacatatcat 7131 175495 Coding 4 1442 caaagttaaagcatcaggtt 79 32 175496 Coding 4 1765ctagtgcttccatcggaagg 37 33 175497 3′UTR 4 3424 aatgccacataccttctaga 2434 175498 5′UTR 4 110 tcgtgagactagagagaagc 71 35 175499 3′UTR 4 3094atgaaaggttactgccttct 81 36 175500 Coding 4 912 tcagcaccaagcaggtcata 8 37175501 3′UTR 4 2841 aagtttgtgcagtattgtag 33 38 175502 Coding 4 2396ctgagcattctgcaaagcta 0 39 175503 Coding 4 350 ttcagattctttacttcgcc 54 40175504 Coding 4 2320 gataacacgttagggcttct 41 41 175505 Coding 4 2331tcaaagcgacagataacacg 51 42 175506 Coding 4 1091 caaagcatgataatattcat 5643 175507 Coding 4 565 ccatcatctgtgagaaccat 86 44 175508 Coding 4 2222atatggtgatgatgtggcac 76 45 175509 5′UTR 4 51 ctcctcaggtggcttgtcag 33 46175510 3′UTR 4 2931 tgagctgtctgtgatccagc 94 47 175511 Coding 4 2321agataacacgttagggcttc 86 48 175512 Start 4 248 catggtgaatcggtccccgc 76 49Codon 175513 Coding 4 1224 tgttatatatgacagttgct 73 50 224184 Coding 4414 ccttatcaagatgcgaactc 63 51 224185 Coding 4 480 ccaaatcaccagcatccaga32 52 224186 Coding 4 619 aactgagttaatcccatgta 72 53 224187 Coding 4 627ttagttcaaactgagttaat 31 54 224188 Coding 4 706 aggccatttctgtgtgtaag 6255 224189 Coding 4 961 ctatctaaaggaatttcaat 10 56 224190 Coding 4 1036cccatcaattcggtaattct 41 57 224191 Coding 4 1125 tatcatgatgagttttggtc 8158 224192 Coding 4 1283 aataataccactcacaacgt 60 59 224193 Coding 4 1380caactttggtgaatagctga 71 60 224194 Coding 4 1699 agtgactctggatttggttc 4461 224195 Coding 4 1928 catctccaagtctaaatctg 36 62 224196 Coding 4 1995ctaatggtgacaactgatcg 72 63 224197 Coding 4 2126 cactgtttttaattcatcag 6564 224198 Coding 4 2457 ataatgttccaattcctact 31 65 224199 Stop 4 2735agaaaaagctcagttaactt 57 66 Codon 224200 3′UTR 4 2828attgtagccaggcttctaaa 68 67 224201 3′UTR 4 3056 atcttcttaaaaataattcg 1868 224202 3′UTR 4 3193 tgtgcaattgtggctaccac 76 69 224203 3′UTR 4 3316aacaatgtcatgttccaggt 88 70 224204 3′UTR 4 3486 gctggcaaagtgactataga 7271 224205 3′UTR 4 3896 ttccacagaagatgtttatt 30 72 224206 3′UTR 4 3899tttttccacagaagatgttt 14 73 224207 intron 11 11258 tagagctaaacgatctagaa47 74 224208 intron 11 23630 taactctttctggccttgaa 93 75 224209 intron 1125682 attggccctaacagaaaatc 19 76 224210 intron: 11 27616agaacttatcctacttaaca 7 77 exon junction 224211 intron 11 39357gtttccctcgtgttgctcag 63 78 224212 exon: 11 39759 ttgtacttactatcatgatg 2579 intron junction 224213 exon: 11 41520 acttacttacctcacaacgt 9 80intron junction 224214 intron: 11 47989 aatctgtgtcctttaaaaca 35 81 exonjunction 224215 exon 11 2745 tgtgcactgaggagctgagg 19 82 224216 exon 4296 acgttcagaacttatctttt 45 83 224217 Stop 13 2221 catgctaaataattcctact0 84 Codon

As shown in Table 1, SEQ ID NOs 14, 15, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 28, 29, 30, 31, 32, 35, 36, 40, 41, 42, 43, 44, 45, 47, 48, 49,50, 51, 53, 55, 57, 58, 59, 60, 61, 63, 64, 66, 67, 69, 70, 71, 74, 75,78 and 83 demonstrated at least 40% inhibition of human HIF1α expressionin this assay and are therefore preferred. More preferred are SEQ ID NOs47, 48 and 25. The target regions to which these preferred sequences arecomplementary are herein referred to as “preferred target segments” andare therefore preferred for targeting by compounds of the presentinvention. These preferred target segments are shown in Table 2. Thesequences represent the reverse complement of the preferred antisensecompounds shown in Table 1. “Target site” indicates the first (5′-most)nucleotide number on the particular target nucleic acid to which theoligonucleotide binds. Also shown in Table 2 is the species in whicheach of the preferred target segments was found.

TABLE 2 Sequence and position of preferred target segments identified inHIF1α. TARGET SITE SEQ ID TARGET REV COMP SEQ ID ID NO SITE SEQUENCE OFSEQ ID ACTIVE IN NO 90592 4 2496 tgcagctactacatcacttt 14 H. sapiens 8590593 4 854 acattcacgtatatgatacc 15 H. sapiens 86 90595 4 2039gcgcaagtcctcaaagcaca 17 H. sapiens 87 90596 4 583 ggtgacatgatttacatttc18 H. sapiens 88 90597 4 1408 gatacaagtagcctctttga 19 H. sapiens 8990598 4 1674 caatcaagaagttgcattaa 20 H. sapiens 90 90599 4 3333gttaatcatataataatgat 21 H. sapiens 91 90600 4 152 cacctctggacttgcctttc22 H. sapiens 92 90601 4 3027 atttttggctagggagttta 23 H. sapiens 9390602 4 2085 acaagaacctactgctaatg 24 H. sapiens 94 90603 4 3101agtaacctttcatcatgatc 25 H. sapiens 95 90604 4 1001 acagcctggatatgaaattt26 H. sapiens 96 90606 4 1983 gttacgttccttcgatcagt 28 H. sapiens 9790607 4 2404 cagaatgctcagagaaagcg 29 H. sapiens 98 90608 4 649ggacacagtgtgtttgattt 30 H. sapiens 99 90609 4 1139 atgatatgtttactaaagga31 H. sapiens 100 90610 4 1442 aacctgatgctttaactttg 32 H. sapiens 10190613 4 110 gcttctctctagtctcacga 35 H. sapiens 102 90614 4 3094agaaggcagtaacctttcat 36 H. sapiens 103 90618 4 350 ggcgaagtaaagaatctgaa40 H. sapiens 104 90619 4 2320 agaagccctaacgtgttatc 41 H. sapiens 10590620 4 2331 cgtgttatctgtcgctttga 42 H. sapiens 106 90621 4 1091atgaatattatcatgctttg 43 H. sapiens 107 90622 4 565 atggttctcacagatgatgg44 H. sapiens 108 90623 4 2222 gtgccacatcatcaccatat 45 H. sapiens 10990625 4 2931 gctggatcacagacagctca 47 H. sapiens 110 90626 4 2321gaagccctaacgtgttatct 48 H. sapiens 111 90627 4 248 gcggggaccgattcaccatg49 H. sapiens 112 90628 4 1224 agcaactgtcatatataaca 50 H. sapiens 113140838 4 414 gagttcgcatcttgataagg 51 H. sapiens 114 140840 4 619tacatgggattaactcagtt 53 H. sapiens 115 140842 4 706 cttacacacagaaatggcct55 H. sapiens 116 140844 4 1036 agaattaccgaattgatggg 57 H. sapiens 117140845 4 1125 gaccaaaactcatcatgata 58 H. sapiens 118 140846 4 1283acgttgtgagtggtattatt 59 H. sapiens 119 140847 4 1380tcagctattcaccaaagttg 60 H. sapiens 120 140848 4 1699gaaccaaatccagagtcact 61 H. sapiens 121 140850 4 1995cgatcagttgtcaccattag 63 H. sapiens 122 140851 4 2126ctgatgaattaaaaacagtg 64 H. sapiens 123 140853 4 2735aagttaactgagctttttct 66 H. sapiens 124 140854 4 2828tttagaagcctggctacaat 67 H. sapiens 125 140856 4 3193gtggtagccacaattgcaca 69 H. sapiens 126 140857 4 3316acctggaacatgacattgtt 70 H. sapiens 127 140858 4 3486tctatagtcactttgccagc 71 H. sapiens 128 140861 11 11258ttctagatcgtttagctcta 74 H. sapiens 129 140862 11 23630ttcaaggccagaaagagtta 75 H. sapiens 130 140865 11 39357ctgagcaacacgagggaaac 78 H. sapiens 131 140870 4 296 aaaagataagttctgaacgt83 H. sapiens 132

As these “preferred target segments” have been found by experimentationto be open to, and accessible for, hybridization with the antisensecompounds of the present invention, one of skill in the art willrecognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother compounds that specifically hybridize to these preferred targetsegments and consequently inhibit the expression of HIF1α.

According to the present invention, antisense compounds includeantisense oligomeric compounds, antisense oligonucleotides, ribozymes,external guide sequence (EGS) oligonucleotides, alternate splicers,primers, probes, and other short oligomeric compounds which hybridize toat least a portion of the target nucleic acid.

Example 16 Western Blot Analysis of HIF1α or HIF2α Prot in Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16–20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to HIF1α or HIF2α isused, with a radiolabeled or fluorescently labeled secondary antibodydirected against the primary antibody species. Bands are visualizedusing a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

Example 17 Additional Antisense Oligonucleotides Against Human HIF1α

A series of antisense compounds were designed to target differentregions of the human HIF1α RNA, using published sequences (GenBankaccession number U29165.1, incorporated herein by reference andincorporated herein as SEQ ID NO: 133). The compounds are shown in Table3. “Target site” indicates the first (5′-most) nucleotide number on theparticular target sequence to which the compound binds. All compounds inTable 3 are chimeric oligonucleotides (“gapmers”) 20 nucleotides inlength, composed of a central “gap” region consisting of ten2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on human HIF1α mRNA levels by quantitative real-time PCR asdescribed in other examples herein. Data are averages from threeexperiments in which A549 cells were treated with the antisenseoligonucleotides of the present invention. “Species” indicates theanimal species of HIF1α nucleic acid to which the compounds are fullycomplementary (H=human, M=mouse, R=rat). As noted many of the compoundsare fully complementary to more than one species.

TABLE 3 Inhibition of human HIF1α mRNA levels by additional chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET seq id TARGET SEQ ISIS # REGION no site Sequence % INHIB ID NOSpecies 298690 Coding 133 373 tgatgagcaagctcataaaa 51 134 H, M, R 298691Coding 133 378 gcaactgatgagcaagctca 77 135 H, M, R 298692 Coding 133 385ggaagtggcaactgatgagc 62 136 H, M, R 298693 Coding 133 631ccagttagttcaaactgagt 79 137 H, M, R 298694 Coding 133 636tgtgtccagttagttcaaac 79 138 H, M, R 298695 Coding 133 641cacactgtgtccagttagtt 79 139 H, M, R 298696 Coding 133 663cacatggatgagtaaaatca 69 140 H, M 298697 Coding 133 673tcctcatggtcacatggatg 84 141 H, M, R 298698 Coding 133 682tctctcatttcctcatggtc 80 142 H, M, R 298699 Coding 133 687gcatttctctcatttcctca 73 143 H, M, R 298700 Coding 133 695gtgtgtaagcatttctctca 67 144 H, M, R 298701 Coding 133 705ggccatttctgtgtgtaagc 78 145 H, M, R 298702 Coding 133 865tggttactgttggtatcata 85 146 H, M 298703 Coding 133 919tcacaaatcagcaccaagca 57 147 H, M, R 298704 Coding 133 924tgggttcacaaatcagcacc 71 148 H, M, R 298705 Coding 133 931tgaggaatgggttcacaaat 69 149 H, M, R 298706 Coding 133 967gtcttgctatctaaaggaat 58 150 H, M 298707 Coding 133 1078tattcataaattgagcggcc 80 151 H, M 298708 Coding 133 1084tgataatattcataaattga 13 152 H, M, R 298709 Coding 133 1117tgagttttggtcagatgatc 64 153 H, M, R 298710 Coding 133 1144acttgtcctttagtaaacat 58 154 H, M, R 298711 Coding 133 1149tggtgacttgtcctttagta 75 155 H, M, R 298712 Coding 133 1154tcctgtggtgacttgtcctt 76 156 H, M, R 298713 Coding 133 1159tactgtcctgtggtgacttg 62 157 H, M, R 298714 Coding 133 1164tcctgtactgtcctgtggtg 83 158 H, M, R 298715 Coding 133 1171gcaagcatcctgtactgtcc 67 159 H, M, R 298716 Coding 133 1192cagacatatccacctctttt 56 160 H, M, R 298717 Coding 133 1198tcaacccagacatatccacc 53 161 H, M, R 298718 Coding 133 1217tatgacagttgcttgagttt 64 162 H, M 298719 Coding 133 1222ttatatatgacagttgcttg 69 163 H, M 298720 Coding 133 1308gaagggagaaaatcaagtcg 46 164 H, M, R 298721 Coding 133 1320attctgtttgttgaagggag 43 165 H, M, R 298722 Coding 133 1354ttcatatctgaagattcaac 53 166 H, M, R 298723 Coding 133 1387tctgattcaactttggtgaa 59 167 H, M 298724 Coding 133 1549attacatcattatataatgg 39 168 H, M 298725 Coding 133 1639ctacttcgaagtggctttgg 77 169 H, M, R 298726 Coding 133 1645tcagcactacttcgaagtgg 80 170 H, M, R 298727 Coding 133 1771ctttgtctagtgcttccatc 73 171 H, M, R 298728 Coding 133 1955atcatccattgggatatagg 74 172 H, M, R 298729 Coding 133 1996tctaatggtgacaactgatc 78 173 H, M, R 298730 Coding 133 2421catcatgttccatttttcgc 69 174 H, M, R 298731 Coding 133 2632gtcagctgtggtaatccact 69 175 H, M, R 298732 Coding 133 2638taactggtcagctgtggtaa 58 176 H, M, R 298733 Coding 133 2659ggagcattaacttcacaatc 39 177 H, M, R 298734 Coding 133 2680aggtttctgctgccttgtat 65 178 H, M, R 298735 Coding 133 2689ccctgcagtaggtttctgct 63 179 H, M, R 298736 Coding 133 2694cttcaccctgcagtaggttt 76 180 H, M, R 298737 Coding 133 2699taattcttcaccctgcagta 71 181 H, M, R 298738 Coding 133 2704ctgagtaattcttcaccctg 77 182 H, M, R 298739 Coding 133 2709aagctctgagtaattcttca 84 183 H, M, R 298740 Coding 133 2714atccaaagctctgagtaatt 66 184 H, M, R 298741 Coding 133 2719acttgatccaaagctctgag 72 185 H, M, R 298742 Stop 133 2728gctcagttaacttgatccaa 80 186 H, M, R codon 298743 3′UTR 133 2770tgagccaccagtgtccaaaa 85 187 H, M, R 298744 3′UTR 133 2821ccaggcttctaaaattagat 68 188 H, M 298745 3′UTR 133 2835gtgcagtattgtagccaggc 78 189 H, M 298746 3′UTR 133 2840agtttgtgcagtattgtagc 74 190 H, M 298747 3′UTR 133 3004taaataaaaaggtgcatttt 0 191 H, M, R 298749 3′UTR 133 3110actgcctatgatcatgatga 74 192 H, M 298750 3′UTR 133 3194ttgtgcaattgtggctacca 79 193 H, M, R 298751 3′UTR 133 3199atatattgtgcaattgtggc 0 194 H, M, R 298752 3′UTR 133 3204agaaaatatattgtgcaatt 31 195 H, M, R 298753 3′UTR 133 3264cttaaaaactagttttataa 21 196 H, M, R 298754 3′UTR 133 3382atgtaaatggctttacccat 68 197 H, M, R 298755 3′UTR 133 3437ttttatccaaataaatgcca 59 198 H, M, R 298756 3′UTR 133 3443tgagaattttatccaaataa 44 199 H, M, R 298757 3′UTR 133 3701taatagcgacaaagtgcata 81 200 H, M, R 298758 3′UTR 133 3706gatgttaatagcgacaaagt 54 201 H, M, R 298759 3′UTR 133 3711aaaaggatgttaatagcgac 77 202 H, M, R 298760 3′UTR 133 3752aatgcttctaaaattactca 62 203 H, M, R 298761 3′UTR 133 3766tatattcctaaaataatgct 30 204 H, M 298762 3′UTR 133 3892acagaagatgtttatttgat 44 205 H, M, RIn Table 3, SEQ ID NO 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 153, 154, 155, 156, 157, 158,159, 160, 161, 162, 163, 166, 167, 169, 170, 171, 172, 173, 174, 175,176, 178, 179, 180, 181, 182, 184, 185, 186, 187, 188, 189, 190, 192,193, 197, 198, 200, 201, 202 and 203 demonstrated at least 50%inhibition of HIF1α expression and are therefore preferred.

Example 18 Antisense Inhibition of Mouse HIF1α Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

In accordance with the present invention, a series of antisensecompounds were designed to target different regions of the mouse HIF1αRNA, using published sequences (GenBank accession number NM_(—)010431.1,incorporated herein by reference and incorporated herein as SEQ ID NO:206. The compounds are shown in Table 4. “Target site” indicates thefirst (5′-most) nucleotide number on the particular target sequence towhich the compound binds. All compounds in Table 4 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on mouseHIF1α mRNA levels by quantitative real-time PCR as described in otherexamples herein. Unlike previous examples, the oligonucleotideconcentration in this experiment is 50 nM. Data are averages from threeexperiments in which b.END cells were treated with the antisenseoligonucleotides of the present invention. In Table 4, “Species”indicates the animal species of HIF1α nucleic acid to which thecompounds are fully complementary (H=human, M=mouse, R=rat). As notedmany of the compounds are fully complementary to more than one species.

TABLE 4 Inhibition of mouse HIF1α mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET TARGET SEQ ISIS # REGION SEQ ID SITE Sequence % INHIB ID NOSpecies 298690 Coding 206 366 tgatgagcaagctcataaaa 32 134 H, M, R 298691Coding 206 371 gcaactgatgagcaagctca 67 135 H, M, R 298692 Coding 206 378ggaagtggcaactgatgagc 33 136 H, M, R 298693 Coding 206 624ccagttagttcaaactgagt 58 137 H, M, R 298694 Coding 206 629tgtgtccagttagttcaaac 39 138 H, M, R 298695 Coding 206 634cacactgtgtccagttagtt 71 139 H, M, R 298696 Coding 206 656cacatggatgagtaaaatca 60 140 H, M 298697 Coding 206 666tcctcatggtcacatggatg 56 141 H, M, R 298698 Coding 206 675tctctcatttcctcatggtc 69 142 H, M, R 298699 Coding 206 680gcatttctctcatttcctca 70 143 H, M, R 298700 Coding 206 688gtgtgtaagcatttctctca 64 144 H, M, R 298701 Coding 206 698ggccatttctgtgtgtaagc 46 145 H, M, R 298702 Coding 206 858tggttactgttggtatcata 69 146 H, M 298703 Coding 206 912tcacaaatcagcaccaagca 45 147 H, M, R 298704 Coding 206 917tgggttcacaaatcagcacc 34 148 H, M, R 298705 Coding 206 924tgaggaatgggttcacaaat 64 149 H, M, R 298706 Coding 206 960gtcttgctatctaaaggaat 42 150 H, M 298707 Coding 206 1071tattcataaattgagcggcc 64 151 H, M 298708 Coding 206 1077tgataatattcataaattga 0 152 H, M, R 298709 Coding 206 1110tgagttttggtcagatgatc 26 153 H, M, R 298710 Coding 206 1137acttgtcctttagtaaacat 47 154 H, M, R 298711 Coding 206 1142tggtgacttgtcctttagta 64 155 H, M, R 298712 Coding 206 1147tcctgtggtgacttgtcctt 58 156 H, M, R 298713 Coding 206 1152tactgtcctgtggtgacttg 48 157 H, M, R 298714 Coding 206 1157tcctgtactgtcctgtggtg 61 158 H, M, R 298715 Coding 206 1164gcaagcatcctgtactgtcc 70 159 H, M, R 298716 Coding 206 1185cagacatatccacctctttt 43 160 H, M, R 298717 Coding 206 1191tcaacccagacatatccacc 55 161 H, M, R 298718 Coding 206 1210tatgacagttgcttgagttt 39 162 H, M 298719 Coding 206 1215ttatatatgacagttgcttg 42 163 H, M 298720 Coding 206 1301gaagggagaaaatcaagtcg 23 164 H, M, R 298721 Coding 206 1313attctgtttgttgaagggag 30 165 H, M, R 298722 Coding 206 1347ttcatatctgaagattcaac 5 166 H, M, R 298723 Coding 206 1380tctgattcaactttggtgaa 52 167 H, M 298724 Coding 206 1542attacatcattatataatgg 29 168 H, M 298725 Coding 206 1629ctacttcgaagtggctttgg 57 169 H, M, R 298726 Coding 206 1635tcagcactacttcgaagtgg 59 170 H, M, R 298727 Coding 206 1761ctttgtctagtgcttccatc 46 171 H, M, R 298728 Coding 206 1987atcatccattgggatatagg 29 172 H, M, R 298729 Coding 206 2028tccaatggtgacaactgatc 19 173 H, M, R 298730 Coding 206 2444catcatgttccatttttcgc 55 174 H, M, R 298731 Coding 206 2655gtcagctgtggtaatccact 59 175 H, M, R 298732 Coding 206 2661taactggtcagctgtggtaa 62 176 H, M, R 298733 Coding 206 2682ggagcattaacttcacaatc 32 177 H, M, R 298734 Coding 206 2703aggtttctgctgccttgtat 50 178 H, M, R 298735 Coding 206 2712ccctgcagtaggtttctgct 53 179 H, M, R 298736 Coding 206 2717cttcaccctgcagtaggttt 46 180 H, M, R 298737 Coding 206 2722taattcttcaccctgcagta 42 181 H, M, R 298738 Coding 206 2727ctgagtaattcttcaccctg 62 182 H, M, R 298739 Coding 206 2732aagctctgagtaattcttca 44 183 H, M, R 298740 Coding 206 2737atccaaagctctgagtaatt 42 184 H, M, R 298741 Coding 206 2742acttgatccaaagctctgag 47 185 H, M, R 298742 Stop 206 2751gctcagttaacttgatccaa 67 186 H, M, R codon 298743 3′UTR 206 2853tgagccaccagtgtccaaaa 56 187 H, M, R 298744 3′UTR 206 2895ccaggcttctaaaattagat 48 188 H, M 298745 3′UTR 206 2909gtgcagtattgtagccaggc 72 189 H, M 298746 3′UTR 206 2914agtttgtgcagtattgtagc 62 190 H, M 298747 3′UTR 206 3067taaataaaaaggtgcatttt 4 191 H, M, R 298748 3′UTR 206 3162gatcatgatgagaatttact 56 207 M 298749 3′UTR 206 3171 actgcctatgatcatgatga64 192 H, M, 298750 3′UTR 206 3253 ttgtgcaattgtggctacca 74 193 H, M, R298751 3′UTR 206 3258 atatattgtgcaattgtggc 67 194 H, M, R 298752 3′UTR206 3263 agaaaatatattgtgcaatt 24 195 H, M, R 298753 3′UTR 206 3322cttaaaaactagttttataa 0 196 H, M, R 298754 3′UTR 206 3428atgtaaatggctttacccat 51 197 H, M, R 298755 3′UTR 206 3483ttttatccaaataaatgcca 28 198 H, M, R 298756 3′UTR 206 3489tgagaattttatccaaataa 14 199 H, M, R 298757 3′UTR 206 3739taatagcgacaaagtgcata 43 200 H, M, R 298758 3′UTR 206 3744gatgttaatagcgacaaagt 23 201 H, M, R 298759 3′UTR 206 3749aaaaggatgttaatagcgac 45 202 H, M, R 298760 3′UTR 206 3789aatgcttctaaaattactca 30 203 H, M, R 298761 3′UTR 206 3803tatattcctaaaataatgct 0 204 H, M 298762 3′UTR 206 3928acagaagatgtttatttgat 21 205 H, M, RIn Table 4, SEQ ID NOs 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 154, 155, 156, 157, 158, 159,160, 161, 162, 163, 167, 169, 170, 171, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 207, 192, 193,194, 197, 200, and 202 demonstrated at least 32% inhibition of HIF1αexpression and are therefore preferred.

Example 19 Real-Time Quantitative PCR Analysis of HIF2α mRNA Levels

Quantitation of HIF2α mRNA levels was accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) asdescribed in previous examples.

Probes and primers to human HIF2α were designed to hybridize to a humanHIF2α sequence, using published sequence information (GenBank accessionnumber NM_(—)001430.1, incorporated herein by reference and incorporatedherein as SEQ ID NO: 208). For human HIF2α the PCR primers were:

forward primer: AAGCCTTGGAGGGTTTCATTG (SEQ ID NO: 209) reverse primer:TGCTGATGTTTTCTGACAGAAAGAT (SEQ ID NO: 210) and the PCR probe was:FAM-CGTGGTGACCCAAGATGGCGACA-TAMRA (SEQ ID NO: 211) where FAM is thefluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCRprimers and probe were those listed in previous examples (SEQ ID NOs: 8,9, 10).

Probes and primers to mouse HIF2α were designed to hybridize to a mouseHIF2α sequence, using published sequence information (GenBank accessionnumber NM_(—)010137.1, incorporated herein by reference and incorporatedherein as SEQ ID NO: 212). For mouse HIF2α the PCR primers were:

forward primer: GGCCATCGTTCGAGCCTTA (SEQ ID NO: 213) reverse primer:GGCACGGGCACGTTCA (SEQ ID NO: 214) and the PCR probe was:FAM-CTGTTGCCGGAACTGACCAGATATGACTG-TAMRA (SEQ ID NO: 215) where FAM isthe fluorescent reporter dye and TAMRA is the quencher dye. For mouseGAPDH the PCR primers were:forward primer: GGCAAATTCAACGGCACAGT(SEQ ID NO: 216) reverse primer:GGGTCTCGCTCCTGGAAGAT(SEQ ID NO: 217) and the PCR probe was:5′JOE-AGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID NO: 218) where JOE isthe fluorescent reporter dye and TAMRA is the quencher dye.

Example 20 Northern Blot Analysis of HIF2α mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Electrophoresis and blotting was performed asdescribed in previous examples.

To detect human HIF2α, a human HIF2α specific probe was prepared by PCRusing the forward primer AAGCCTTGGAGGGTTTCATTG (SEQ ID NO: 209) and thereverse primer TGCTGATGTTTTCTGACAGAAAGAT (SEQ ID NO: 210). To normalizefor variations in loading and transfer efficiency membranes werestripped and probed for human glyceraldehyde-3-phosphate dehydrogenase(GAPDH) RNA (Clontech, Palo Alto, Calif.).

To detect mouse HIF2α, a mouse HIF2α specific probe was prepared by PCRusing the forward primer GGCCATCGTTCGAGCCTTA (ID NO: 213) and thereverse primer GGCACGGGCACGTTCA (SEQ ID NO: 214). To normalize forvariations in loading and transfer efficiency membranes were strippedand probed for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH)RNA (Clontech, Palo Alto, Calif.).

Example 21 Antisense Inhibition of Human HIF2α Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

In accordance with the present invention, a series of antisensecompounds were designed to target different regions of the human HIF2αRNA, using published sequences (GenBank accession number NM_(—)001430.1,incorporated herein by reference and incorporated herein as SEQ ID NO:208). The compounds are shown in Table 5. “Target site” indicates thefirst (5′-most) nucleotide number on the particular target sequence towhich the compound binds. All compounds in Table 5 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on humanHIF2α mRNA levels by quantitative real-time PCR as described in otherexamples herein. Data are averages from three experiments in which A549cells were treated with the antisense oligonucleotides of the presentinvention.

TABLE 5 Inhibition of human HIF2α mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET SEQ ID TARGET % SEQ ID ISIS # REGION NO SITE SEQUENCE INHIB NO221985 Start 208 142 gtcagctgtcattgtcgctg 74 219 Codon 221987 Stop 2082751 ggcctggctcaggtggcctg 54 220 Codon 221989 Coding 208 1000ggtcatgttctcggagtcta 82 221 221991 Coding 208 1572 gtggagcagctgctgctgct80 222 221993 Coding 208 2412 ggtacatttgcgctcagtgg 76 223 221995 Coding208 2206 tgggcctcgagccccaaaac 15 224 221997 Coding 208 1300gaataggaagttactcttct 51 225 221999 Coding 208 1752 tggaagtcttccccgtccat69 226 222001 Coding 208 947 gcagctcctcagggtggtaa 82 227 222003 Coding208 977 catggtagaattcataggct 82 228 222005 Coding 208 1631tcacttcaatcttcaggtcg 55 229 222007 Coding 208 2691 gagcttcccagcacgggcac79 230 222009 Coding 208 1502 tgaaggcaggcaggctccca 77 231 222011 Coding208 2008 ggtgctggcctggccacagc 72 232 222013 Coding 208 561cgaatctcctcatggtcgca 89 233 222015 Coding 208 1247 tgctgttcatggccatcagg78 234 222017 Coding 208 1679 tactgcattggtccttggcc 78 235 222019 Coding208 1488 ctcccagcctcgctctgggt 63 236 222021 Coding 208 2700aggagcgtggagcttcccag 59 237 222023 Coding 208 623 ctgtggacatgtctttgctt79 238 222025 Coding 208 1716 agtgtctccaagtccagctc 84 239 222027 Coding208 759 ctattgtgaggagggcagtt 75 240 222029 Coding 208 237tcatagaacacctccgtctc 37 241 222031 Coding 208 2334 aaatgtgaggtgctgccacc67 242 222033 Coding 208 1578 ttgggcgtggagcagctgct 54 243 222035 Coding208 2126 gcgctgctcccaagaactct 89 244 222037 Coding 208 2639gcagcaggtaggactcaaat 64 245 222039 Coding 208 2325 gtgctgccaccaggtgggtc79 246 222041 Coding 208 1001 tggtcatgttctcggagtct 82 247 222043 Coding208 1209 tcagtctggtccatggagaa 80 248 222045 Coding 208 566tctcacgaatctcctcatgg 68 249 222047 Coding 208 1622 tcttcaggtcgttatccaaa56 250 222049 Coding 208 2715 aggtcccctccttgcaggag 66 251 222051 Coding208 246 tgggccagctcatagaacac 82 252 222053 Coding 208 2336tcaaatgtgaggtgctgcca 73 253 222055 Coding 208 391 catctgctggtcagcttcgg85 254 222057 Coding 208 1217 acagggattcagtctggtcc 84 255As shown in Table 5, SEQ ID NOs 219, 220, 221, 211, 223, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 242,243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254 and 255demonstrated at least 40% inhibition of HIF2α expression and aretherefore preferred. More preferred are SEQ ID NOs 233, 239 and 244. Thetarget regions to which these preferred sequences are complementary areherein referred to as “preferred target segments” and are thereforepreferred for targeting by compounds of the present invention. Thesepreferred target segments are shown in Table 7. These sequences areshown to contain thymine (T) but one of skill in the art will appreciatethat thymine (T) is generally replaced by uracil (U) in RNA sequences.The sequences represent the reverse complement of the preferredantisense compounds shown in Table 5. “Target site” indicates the first(5′-most) nucleotide number on the particular target nucleic acid towhich the oligonucleotide binds. Also shown in Table 7 is the species inwhich each of the preferred target segments was found.

Example 22 Antisense Inhibition of Mouse HIF2α Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

In accordance with the present invention, a second series of antisensecompounds were designed to target different regions of the mouse HIF2αRNA, using published sequences (GenBank accession number NM_(—)010137.1,incorporated herein by reference and incorporated herein as SEQ ID NO:212, nucleotides 20468925 to 20547619 of the sequence with GenBankaccession number NW_(—)000133.1, incorporated herein by reference andincorporated herein as SEQ ID NO: 257, GenBank accession numberBY229956.1, incorporated herein by reference and incorporated herein asSEQ ID NO: 258, and GenBank accession number AK087208.1, incorporatedherein by reference and incorporated herein as SEQ ID NO: 259). Thecompounds are shown in Table 6. “Target site” indicates the first(5′-most) nucleotide number on the particular target nucleic acid towhich the compound binds. All compounds in Table 6 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′-directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on mouseHIF2α mRNA levels by quantitative real-time PCR as described in otherexamples herein. Data are averages from three experiments in which b.ENDcells were treated with the antisense oligonucleotides of the presentinvention.

TABLE 6 Inhibition of mouse HIF2α mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET SEQ ID TARGET % SEQ ISIS # REGION NO: SITE SEQUENCE INHIB ID NO320972 5′UTR 212 130 ggttccttaaccccgtaggg 70 260 320973 5′UTR 212 135acctgggttccttaaccccg 61 261 320974 5′UTR 212 140 ggagcacctgggttccttaa 70262 320975 Start Codon 212 178 ttgtcagctgtcattgtcgc 72 263 320976 StartCodon 212 183 tctccttgtcagctgtcatt 84 264 320977 Coding 212 266gaagacctccgtctccttgc 83 265 320978 Coding 212 317 caggtgggagctcacactgt76 266 320979 Coding 212 352 aagctgatggccaggcgcat 64 267 320980 Coding212 442 ttcaggtacaagttatccat 78 268 320981 Coding 212 448aaggctttcaggtacaagtt 73 269 320982 Coding 212 461 aatgaaaccctccaaggctt87 270 320983 Coding 212 520 atgaacttgctgatgttttc 29 271 320984 Coding212 525 gtcccatgaacttgctgatg 57 272 320985 Coding 212 535acctgggtaagtcccatgaa 63 273 320986 Coding 212 545 tgttagttctacctgggtaa62 274 320987 Coding 212 563 gtcaaagatgctgtgtcctg 83 275 320988 Coding212 574 ggatgagtgaagtcaaagat 50 276 320989 Coding 212 673atgaagaagtcacgctcggt 63 277 320990 Coding 212 682 ttcatcctcatgaagaagtc53 278 320991 Coding 212 687 tgcacttcatcctcatgaag 58 279 320992 Coding212 714 tgacagtccggcctctgttg 52 280 320993 Coding 212 766actctcacttgcccggtgca 87 281 320994 Coding 212 776 gttgttgtagactctcactt64 282 320995 Coding 212 850 attggctcacacatgatgat 76 283 320996 Coding212 860 tgggtgctggattggctcac 75 284 320997 Coding 212 913atgctgtggcggctcaggaa 87 285 320998 Coding 212 970 gggtggtaaccaatcagttc76 286 320999 Coding 212 1057 gtgcacaagttctggtgact 50 287 321000 Coding212 1062 ccttggtgcacaagttctgg 74 288 321001 Coding 212 1135gtcccctgggtctccagcca 78 289 321002 Coding 212 1140 tgaccgtcccctgggtctcc63 290 321003 Coding 212 1145 gtagatgaccgtcccctggg 68 291 321004 Coding212 1150 gggttgtagatgaccgtccc 62 292 321005 Coding 212 1191catagttgacacacatgata 37 293 321006 Coding 212 1234 tccatggagaacaccacgtc76 294 321007 Coding 212 1239 tctggtccatggagaacacc 83 295 321008 Coding212 1286 aaagatgctgttcatggcca 51 296 321009 Coding 212 1338tggtgaacaggtagttgctc 64 297 321010 Coding 212 1363 agctcctcgggctcctcctt83 298 321011 Coding 212 1454 ggccttgccataggctgagg 49 299 321012 Coding212 1459 aggatggccttgccataggc 53 300 321013 Coding 212 1612ctgctgggcgtggagcagct 40 301 321014 Coding 212 1725 tgaagtccgtctgggtactg58 302 321015 Coding 212 1939 tccaactgctgcgggtactt 82 303 321016 Coding212 2002 ttgctcccagcatcaaagaa 0 304 321017 Coding 212 2012cagggaccctttgctcccag 81 305 321018 Coding 212 2038 gtgctggcctggccacagca66 306 321019 Coding 212 2216 cttgaacatggagacatgag 65 307 321020 Coding212 2226 cagacctcatcttgaacatg 72 308 321021 Coding 212 2231ctttgcagacctcatcttga 73 309 321022 Coding 212 2296 ttcagcttgttggacagggc51 310 321023 Coding 212 2376 gtgaactgctggtgcctgga 79 311 321024 Coding212 2386 cacatcaagtgtgaactgct 0 312 321025 Coding 212 2413ccgcccatgaggctcttcat 70 313 321026 Coding 212 2423 aggacaggtcccgcccatga85 314 321027 Coding 212 2433 caggcatcaaaggacaggtc 55 315 321028 Coding212 2482 gatttttgggtgaattcatc 38 316 321029 Coding 212 2647ctggccacgcctgacacctt 65 317 321030 Coding 212 2665 gatggccccagcagtcgact64 318 321031 Coding 212 2670 cgaacgatggccccagcagt 48 319 321032 Coding212 2680 aggtaaggctcgaacgatgg 65 320 321033 Coding 212 2707cagtcatatctggtcagttc 78 321 321034 Coding 212 2712 cctcacagtcatatctggtc83 322 321035 Coding 212 2717 gttcacctcacagtcatatc 66 323 321036 Coding212 2722 ggcacgttcacctcacagtc 81 324 321037 Coding 212 2727gcacgggcacgttcacctca 90 325 321038 Coding 212 2758 tctctcccctgcaggagtgt79 326 321039 Coding 212 2768 tctgagaaggtctctcccct 51 327 321040 Coding212 2778 ggtccagagctctgagaagg 73 328 321041 Stop Codon 212 2791gctcaggtggcctggtccag 69 329 321042 Stop Codon 212 2798ggccctggctcaggtggcct 12 330 321043 3′UTR 212 3199 agaacaagaacacttgagtt66 331 321044 intron 257 12633 aacagttgagacatgacagt 67 332 321045 exon:257 74580 tgtcactaacctcatcttga 45 333 intron junction 321046 5′UTR 258235 acaggagtcacttttctggg 43 334 321047 5′UTR 258 82 catacagtctcaggacactg47 335 321048 Genomic 259 116 aatctgtccatgaaaagaca 33 336

As shown in Table 6, SEQ ID NO, 260, 261, 262, 263, 264, 265, 266, 267,268, 269, 270, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 294, 295, 296, 297,298, 299, 300, 301, 302, 303, 305, 306, 307, 308, 309, 310, 311, 313,314, 315, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 331, 332, 333, 334 and 335 demonstrated at least 40% inhibition ofmouse HIF2α expression in this experiment and are therefore preferred.More preferred are SEQ ID NOs 270, 281 and 285. The target regions towhich these preferred sequences are complementary are herein referred toas “preferred target segments” and are therefore preferred for targetingby compounds of the present invention. These preferred target segmentsare shown in Table 7. These sequences are shown to contain thymine (T)but one of skill in the art will appreciate that thymine (T) isgenerally replaced by uracil (U) in RNA sequences. The sequencesrepresent the reverse complement of the preferred antisense compoundsshown in Tables 5 and 6. “Target site” indicates the first (5′-most)nucleotide number on the particular target nucleic acid to which theoligonucleotide binds. Also shown in Table 3 is the species in whicheach of the preferred target segments was found.

TABLE 7 Sequence and position of preferred target segments identified inhypoxia-inducible factor 2 alpha. TARGET SITE SEQ ID TARGET REV COMP SEQID ID NO SITE SEQUENCE OF SEQ ID ACTIVE IN NO 138730 208 142cagcgacaatgacagctgac 291 H. sapiens 337 138731 208 2751caggccacctgagccaggcc 292 H. sapiens 338 138732 208 1000tagactccgagaacatgacc 293 H. sapiens 339 138733 208 1572agcagcagcagctgctccac 294 H. sapiens 340 138734 208 2412ccactgagcgcaaatgtacc 295 H. sapiens 341 138736 208 1300agaagagtaacttcctattc 297 H. sapiens 342 138737 208 1752atggacggggaagacttcca 298 H. sapiens 343 138738 208 947ttaccaccctgaggagctgc 299 H. sapiens 344 138739 208 977agcctatgaattctaccatg 300 H. sapiens 345 138740 208 1631cgacctgaagattgaagtga 301 H. sapiens 346 138741 208 2691gtgcccgtgctgggaagctc 302 H. sapiens 347 138742 208 1502tgggagcctgcctgccttca 303 H. sapiens 348 138743 208 2008gctgtggccaggccagcacc 304 H. sapiens 349 138744 208 561tgcgaccatgaggagattcg 305 H. sapiens 350 138745 208 1247cctgatggccatgaacagca 306 H. sapiens 351 138746 208 1679ggccaaggaccaatgcagta 307 H. sapiens 352 138747 208 1488acccagagcgaggctgggag 308 H. sapiens 353 138748 208 2700ctgggaagctccacgctcct 309 H. sapiens 354 138749 208 623aagcaaagacatgtccacag 310 H. sapiens 355 138750 208 1716gagctggacttggagacact 311 H. sapiens 356 138751 208 759aactgccctcctcacaatag 312 H. sapiens 357 138753 208 2334ggtggcagcacctcacattt 314 H. sapiens 358 138754 208 1578agcagctgctccacgcccaa 315 H. sapiens 359 138755 208 2126agagttcttgggagcagcgc 316 H. sapiens 360 138756 208 2639atttgagtcctacctgctgc 317 H. sapiens 361 138757 208 2325gacccacctggtggcagcac 318 H. sapiens 362 138758 208 1001agactccgagaacatgacca 319 H. sapiens 363 138759 208 1209ttctccatggaccagactga 320 H. sapiens 364 138760 208 566ccatgaggagattcgtgaga 321 H. sapiens 365 138761 208 1622tttggataacgacctgaaga 322 H. sapiens 366 138762 208 2715ctcctgcaaggaggggacct 323 H. sapiens 367 138763 208 246gtgttctatgagctggccca 324 H. sapiens 368 138764 208 2336tggcagcacctcacatttga 325 H. sapiens 369 138765 208 391ccgaagctgaccagcagatg 326 H. sapiens 370 138766 208 1217ggaccagactgaatccctgt 327 H. sapiens 371 237138 212 130ccctacggggttaaggaacc 332 M. musculus 372 237139 212 135cggggttaaggaacccaggt 333 M. musculus 373 237140 212 140ttaaggaacccaggtgctcc 334 M. musculus 374 237141 212 178gcgacaatgacagctgacaa 335 M. musculus 375 237142 212 183aatgacagctgacaaggaga 336 M. musculus 376 237143 212 266gcaaggagacggaggtcttc 337 M. musculus 377 237144 212 317acagtgtgagctcccacctg 338 M. musculus 378 237145 212 352atgcgcctggccatcagctt 339 M. musculus 379 237146 212 442atggataacttgtacctgaa 340 M. musculus 380 237147 212 448aacttgtacctgaaagcctt 341 M. musculus 381 237148 212 461aagccttggagggtttcatt 342 M. musculus 382 237150 212 525catcagcaagttcatgggac 344 M. musculus 383 237151 212 535ttcatgggacttacccaggt 345 M. musculus 384 237152 212 545ttacccaggtagaactaaca 346 M. musculus 385 237153 212 563caggacacagcatctttgac 347 M. musculus 386 237154 212 574atctttgacttcactcatcc 348 M. musculus 387 237155 212 673accgagcgtgacttcttcat 349 M. musculus 388 237156 212 682gacttcttcatgaggatgaa 350 M. musculus 389 237157 212 687cttcatgaggatgaagtgca 351 M. musculus 390 237158 212 714caacagaggccggactgtca 352 M. musculus 391 237159 212 766tgcaccgggcaagtgagagt 353 M. musculus 392 237160 212 776aagtgagagtctacaacaac 354 M. musculus 393 237161 212 850atcatcatgtgtgagccaat 355 M. musculus 394 237162 212 860gtgagccaatccagcaccca 356 M. musculus 395 237163 212 913ttcctgagccgccacagcat 357 M. musculus 396 237164 212 970gaactgattggttaccaccc 358 M. musculus 397 237165 212 1057agtcaccagaacttgtgcac 359 M. musculus 398 237166 212 1062ccagaacttgtgcaccaagg 360 M. musculus 399 237167 212 1135tggctggagacccaggggac 361 M. musculus 400 237168 212 1140ggagacccaggggacggtca 362 M. musculus 401 237169 212 1145cccaggggacggtcatctac 363 M. musculus 402 237170 212 1150gggacggtcatctacaaccc 364 M. musculus 403 237172 212 1234gacgtggtgttctccatgga 366 M. musculus 404 237173 212 1239ggtgttctccatggaccaga 367 M. musculus 405 237174 212 1286tggccatgaacagcatcttt 368 M. musculus 406 237175 212 1338gagcaactacctgttcacca 369 M. musculus 407 237176 212 1363aaggaggagcccgaggagct 370 M. musculus 408 237177 212 1454cctcagcctatggcaaggcc 371 M. musculus 409 237178 212 1459gcctatggcaaggccatcct 372 M. musculus 410 237179 212 1612agctgctccacgcccagcag 373 M. musculus 411 237180 212 1725cagtacccagacggacttca 374 M. musculus 412 237181 212 1939aagtacccgcagcagttgga 375 M. musculus 413 237183 212 2012ctgggagcaaagggtccctg 377 M. musculus 414 237184 212 2038tgctgtggccaggccagcac 378 M. musculus 415 237185 212 2216ctcatgtctccatgttcaag 379 M. musculus 416 237186 212 2226catgttcaagatgaggtctg 380 M. musculus 417 237187 212 2231tcaagatgaggtctgcaaag 381 M. musculus 418 237188 212 2296gccctgtccaacaagctgaa 382 M. musculus 419 237189 212 2376tccaggcaccagcagttcac 383 M. musculus 420 237191 212 2413atgaagagcctcatgggcgg 385 M. musculus 421 237192 212 2423tcatgggcgggacctgtcct 386 M. musculus 422 237193 212 2433gacctgtcctttgatgcctg 387 M. musculus 423 237195 212 2647aaggtgtcaggcgtggccag 389 M. musculus 424 237196 212 2665agtcgactgctggggccatc 390 M. musculus 425 237197 212 2670actgctggggccatcgttcg 391 M. musculus 426 237198 212 2680ccatcgttcgagccttacct 392 M. musculus 427 237199 212 2707gaactgaccagatatgactg 393 M. musculus 428 237200 212 2712gaccagatatgactgtgagg 394 M. musculus 429 237201 212 2717gatatgactgtgaggtgaac 395 M. musculus 430 237202 212 2722gactgtgaggtgaacgtgcc 396 M. musculus 431 237203 212 2727tgaggtgaacgtgcccgtgc 397 M. musculus 432 237204 212 2758acactcctgcaggggagaga 398 M. musculus 433 237205 212 2768aggggagagaccttctcaga 399 M. musculus 434 237206 212 2778ccttctcagagctctggacc 400 M. musculus 435 237207 212 2791ctggaccaggccacctgagc 401 M. musculus 436 237209 212 3199aactcaagtgttcttgttct 403 M. musculus 437 237210 257 12633actgtcatgtctcaactgtt 404 M. musculus 438 237211 257 74580tcaagatgaggttagtgaca 405 M. musculus 439 237212 258 235cccagaaaagtgactcctgt 406 M. musculus 440 237213 258 82cagtgtcctgagactgtatg 407 M. musculus 441

As these “preferred target segments” have been found by experimentationto be open to, and accessible for, hybridization with the antisensecompounds of the present invention, one of skill in the art willrecognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother compounds that specifically hybridize to these preferred targetsegments and consequently inhibit the expression of HIF2α.

According to the present invention, antisense compounds includeantisense oligomeric compounds, antisense oligonucleotides, ribozymes,external guide sequence (EGS) oligonucleotides, alternate splicers,primers, probes, and other short oligomeric compounds which hybridize toat least a portion of the target nucleic acid.

Example 23 Expression of HIF1α and HIF2α in Various Human Cell Lines

U87-MG human glioblastoma, PC-3 human prostate cancer, JEG-3 humanchoriocarcinoma, HeLa human cervix cancer, SK-N-BE(2) neuroblastoma,MCF-7 human breast cancer, 786-O human clear-cell renal cell carcinoma,Calu-1 human lung cancer, and Hep3B human hepatocellular carcinoma cellswere purchased from American Type Culture Collection (ATCC; Manassas,Va.) and cultured according to ATCC directions. Human umbilicalendothelial cells (HUVEC) were obtained from Cascade Biologics (PortlandOreg.). Hypoxic treatments of cells (0.5–0.8×10⁶/60 mm dish or1–2×10⁶/100 mm dish) were performed at 1% O₂ in a chamber controlled byProOx oxygen sensor (BioSpherix, Redfield, N.Y.) for 16 h. To achievethe optimal hypoxic induction, 3 or 6 ml of medium was used for 60 mmand 100 mm dish culture, respectively during incubation. CoCl₂ (150 μM)was added to the cells to mimic hypoxic condition in some experiments.

Cultured cells at normoxia, hypoxia, or with CoCl₂ were harvested andwhole cell lysates prepared with RIPA buffer containing proteaseinhibitor cocktails (Roche), 0.5 mM sodium orthovanadate, 10 mMβ-glycerophophate, 250 ng/ml ubiquitin aldehyde, and 400 nM epoxomicinwere separated on 12% SDS-PAGE and transferred to PVDF membranes(Amersham Biosciences). Typically, 35–50 μg of proteins were loaded perlane. Immunoblotting was performed with the following antibodies:anti-HIF-1α (BD Transduction Laboratories) at 1:250 (v/v); anti-HIF-2α(EPAS1) (Santa Cruz Biotechnology Inc) at 1:150; anti-HIF-1β (BDTransduction Laboratories) at 1:1000; anti-VHL (BD TransductionLaboratories) at 1:500; anti-GLUT-1 (Alpha Diagnostic International) at1:600, and anti-α-tubulin (Sigma) at 1:2000 in 0.05%Tween-20/Tris-buffered saline (T-TBS) blocking buffer containing 5%nonfat skim milk at 4° C. overnight, followed by washing with T-TBS for30 min. Goat anti-mouse or rabbit IgGs coupled with HRP (BioRad) wereused as secondary antibodies at 1:3000. Immunospecific bands weredetected by enhanced chemiluminescence plus (ECL-Plus) detection kit(Amersham Biosciences).

Hif1α expression was shown to be increased in hypoxic conditions and inthe presence of CoCl₂ (which mimics hypoxia) in U87-MG humanglioblastoma, PC-3 human prostate cancer, JEG-3 human choriocarcinoma,HeLa human cervix cancer, SK-N-BE(2) neuroblastoma, MCF-7 human breastcancer, Calu-1 human lung cancer, and Hep3B human hepatocellularcarcinoma cells but not 786-O human clear-cell renal cell carcinomacells.

Hif2α expression was shown to be increased in hypoxic (1% O₂) conditionsand in the presence of CoCl₂ in U87-MG human glioblastoma, PC-3 humanprostate cancer, JEG-3 human choriocarcinoma, MCF-7 human breast cancer,786-O human clear-cell renal cell carcinoma, Calu-1 human lung cancer,Hep3B human hepatocellular carcinoma cells and HUVECs.

Example 24 Antisense Modulation of HIF1α mRNA Expression in Cancer Cells(Dose Response)

HeLa, Hep3B, or U87-MG cells were plated in 96-well plates(8–10,000/well) 16 h prior to transfection. The following antisenseoligonucleotides were delivered into cells by lipofectin (3 μg/ml per100 nM oligonucleotide) in Opti-Mem media (Invitrogen) at the indicatedconcentration: ISIS 175510 (SEQ ID NO: 47) and ISIS 298697 (SEQ ID NO:141) are targeted to human HIF-1α ASOs; ISIS 222035 (SEQ ID NO: 244) istargeted to human HIF-2α; and ISIS 129688 (TTCGCGGCTGGACGATTCAG; SEQ IDNO: 442) is an unrelated control. 10/35 is an equal mixture of ISIS175510 and 222035 (HIF1α and HIF2α inhibitory oligonucleotides). ISIS97/35 is an equal mixture of ISIS 298697 and ISIS 222035 (HIF1α andHIF2α inhibitory oligonucleotides).

The transfection medium was switched to complete growth medium (120μl/well) 4 h after transfection. Sixty microliters of medium was removedfrom the well 3 h after media switch and the cells were furtherincubated at normoxia or hypoxia for 16–20 h.

TABLE 8 HIF1α mRNA expression in antisense treated HeLa cells Shown aspercent inhibition relative to control oligonucleotide Percentinhibition of HIF1α mRNA Oligo- Normoxia expression after treatment withnucleotide or oligonucleotide at concentrations shown: and conditions:Hypoxia 0 6.25 nM 25 nM 100 nM 200 nM 129688 N 0 0 0 30 47 129688 H 1 110 30 57 175510 N 0 24 77 94 94 175510 H 1 39 82 95 96 298697 N 0 44 7291 93 298697 H 3 30 75 92 93 222035 N 0 0 0 1 24 222035 H 3 3 0 11 3510/35 N 0 33 82 94 94 10/35 H 3 35 85 94 95 97/35 N 0 16 66 84 85 97/35H 3 34 79 88 89 N = Normoxia (21% O₂) H = Hypoxia (1% O₂)It can be seen that the HIF1α antisense oligonucleotides ISIS 175510 and298697 specifically inhibited HIF1α and not HIF2α. Similar results wereobtained in Hep3b human hepatocellular carcinoma cells and in U87-MGhuman glioblastoma cells.

Example 25 Antisense Modulation of HIF2α mRNA expression in cancer cells(Dose Response)

HeLa, Hep3B, or U87-MG cells were plated in 96-well plates(8–10,000/well) 16 h prior to transfection. The following antisenseoligonucleotides were delivered into cells by lipofectin (3 μg/ml per100 nM oligonucleotide) in Opti-Mem media (Invitrogen) at the indicatedconcentration: ISIS (SEQ ID NO: 47) and ISIS 298697 (SEQ ID NO: 141) aretargeted to human HIF-1α ASOs; ISIS 222035 (SEQ ID NO: 244) is targetedto human HIF-2α; and ISIS 129688 (TTCGCGGCTGGACGATTCAG; SEQ ID NO: 442)is an unrelated control. 10/35 is an equal mixture of ISIS 175510 and222035 (HIF1α and HIF2α inhibitory oligonucleotides). ISIS 97/35 is anequal mixture of ISIS 298697 and ISIS 222035 (HIF1α and HIF2α inhibitoryoligonucleotides). The transfection medium was switched to completegrowth medium (120 μl/well) 4 h after transfection. Sixty microliters ofmedium was removed from the well 3 h after media switch and the cellswere further incubated at normoxia or hypoxia for 16–20 h.

TABLE 9 HIF2α mRNA expression in ASO treated HeLa cells Shown as percentinhibition relative to control oligonucleotide Percent inhibition ofHIF1α mRNA Oligo- Normoxia expression after treatment with nucleotide oroligonucleotide at concentrations shown: and conditions: Hypoxia 0 6.25nM 25 nM 100 nM 200 nM 129688 N 0 0 16 12 21 129688 H 0 0 4 12 50 175510N 0 1 0 0 0 175510 H 0 8 0 4 0 298697 N 0 0 10 48 65 298697 H 0 0 11 5258 222035 N 0 0 62 93 96 222035 H 0 19 73 94 96 10/35 N 0 0 77 96 9610/35 H 0 21 78 94 95 97/35 N 0 0 63 89 95 97/35 H 0 34 79 96 96 N =Normoxia (21% O₂) H = Hypoxia (1% O₂)It can be seen that the HIF2α antisense oligonucleotide ISIS 222035specifically inhibited HIF2α relative to HIF1α. The oligonucleotide ISIS298697, designed to target human HIF1α, showed some ability to inhibitHIF2α expression as well. This oligonucleotide has perfectcomplementarity to the HIF1α target sequence and was found to have onlytwo mismatches to the human HIF2α. Similar results were seen in U87-MGhuman glioblastoma cells and HepG3 hepatocellular carcinoma cells.

Example 26 HIF2α Plays a Major Role in the Induction of VEGF by Hypoxiain U87-MG Cells

Genes whose products are dramatically induced by hypoxia (or CoCl₂, amimic of hypoxia) include erythropoietin (Epo), glucose transporter-1(Glut-1), vascular endothelial growth factor (VEGF) andPhosphofructokinase-L (PFK-L). They are induced by hypoxia to varyingextents in various cell lines. As shown in previous examples, VEGFexpression is induced by hypoxia in U87-MG cells. The followingantisense oligonucleotides were delivered into cells by lipofectin (3μg/ml per 100 nM oligonucleotide) in Opti-Mem media (Invitrogen) at theindicated concentration: ISIS 175510 (SEQ ID NO: 47) and ISIS 298697(SEQ ID NO: 141) are targeted to human HIF-1α ASOs; ISIS 222035 (SEQ IDNO: 244) is targeted to human HIF-2α; and ISIS 129688(TTCGCGGCTGGACGATTCAG; SEQ ID NO: 442) is an unrelated control. 10/35 isan equal mixture of ISIS 175510 and 222035 (HIF1α and HIF2α inhibitoryoligonucleotides). ISIS 97/35 is an equal mixture of ISIS 298697 andISIS 222035 (HIF1α and HIF2α inhibitory oligonucleotides).

TABLE 10 HIF2α plays a major role in the induction of VEGF by hypoxia inU87-MG cells Oligo- Relative VEGF nucleotide Normoxia mRNA expressionafter treatment with and or oligonucleotide at concentrations shown:conditions: Hypoxia 0 6.25 nM 25 nM 100 nM 200 nM 129688 N 100 103 11173 81 129688 H 372 378 346 363 383 175510 N 100 86 65 61 62 175510 H 372397 407 338 392 298697 N 100 111 81 56 73 298697 H 372 413 342 312 275222035 N 100 94 69 48 45 222035 H 372 399 257 131 108 10/35 N 100 81 4845 44 10/35 H 372 431 254 110 80 97/35 N 100 119 63 45 47 97/35 H 372409 289 124 85ISIS 175510, which specifically inhibits HIF1α and not HIF2α, was foundto have no effect on VEGF induction by hypoxia in U87-MG cells. Incontrast, ISIS 222035, which specifically inhibits HIF2α and not HIF1α,caused a dose-dependent decrease in VEGF induction. ISIS 298697, whichwas designed to target HIF1α but was found to have crossreactivity withHIF2α, also interfered with VEGF induction by hypoxia. Thus HIF2α playsa major role in the induction of VEGF by hypoxia in U87-MG cells.

Example 27 HIF2α Plays a Major Role in the Induction of Epo by Hypoxiain Hep3B Cells

Genes whose products are dramatically induced by hypoxia (or CoCl₂, amimic of hypoxia) include Epo, Glut-1, VEGF and PFK-L. They are inducedby hypoxia to varying extents in various cell lines. Epo(erythropoietin) expression is induced by hypoxia in Hep3B cells. Thefollowing antisense oligonucleotides were delivered into Hep3B cells bylipofectin (3 μg/ml per 100 nM oligonucleotide) in Opti-Mem media(Invitrogen) at the indicated concentration: ISIS 175510 (SEQ ID NO: 47)and ISIS 298697 (SEQ ID NO: 141) are targeted to human HIF-1α ASOs; ISIS222035 (SEQ ID NO: 244) is targeted to human HIF-2α; and ISIS 129688(TTCGCGGCTGGACGATTCAG; SEQ ID NO: 442) is an unrelated control. 10/35 isan equal mixture of ISIS 175510 and 222035 (HIF1α and HIF2α inhibitoryoligonucleotides). ISIS 97/35 is an equal mixture of ISIS 298697 andISIS 222035 (HIF1α HIF2α inhibitory oligonucleotides).

TABLE 11 HIF2α plays a major role in the induction of Epo by hypoxia inHep3B cells Relative Epo mRNA expression after treatment witholigonucleotide at concentrations shown: Normoxia Shown as -Foldinduction over control Oligonucleotide or 6.25 and conditions: Hypoxia 0nM 25 nM 100 nM 200 nM 129688 N 1 1 0 3 11 129688 H 531 586 433 261 128175510 N 1 8 3 3 2 175510 H 531 577 542 326 144 298697 N 1 9 11 12 3298697 H 531 436 326 52 6 222035 N 1 3 3 2 1 222035 H 531 302 101 2 210/35 N 1 2 0 0 3 10/35 H 531 212 30 0 1 97/35 N 1 2 0 1 4 97/35 H 531194 29 2 1ISIS 175510, which specifically inhibits HIF1● and not HIF2●, was foundto have no effect on Epo induction by hypoxia in Hep3B cells. Incontrast, ISIS 222035, which specifically inhibits HIF2● and not HIF1●,caused a dose-dependent decrease in Epo induction. ISIS 298697, whichwas designed to target HIF1● but was found to have crossreactivity withHIF2●, also interfered with Epo induction by hypoxia. Thus HIF2a plays amajor role in the induction of Epo by hypoxia in Hep3B cells.

Example 28 Both HIF1α and HIF2α Play a Major Role in the Induction ofVEGF by Hypoxia in HeLa Cells

Genes whose products are dramatically induced by hypoxia (or CoCl₂)include Epo (erythropoietin), Glut-1, VEGF and Phosphofructokinase(PFK)-L. They are induced by hypoxia to varying extents in various celllines. VEGF expression is induced by hypoxia in HeLa cells. Thefollowing antisense oligonucleotides were delivered into cells bylipofectin (3 μg/ml per 100 nM oligonucleotide) in Opti-Mem media(Invitrogen) at the indicated concentration: ISIS 175510 (SEQ ID NO: 47)and ISIS 298697 (SEQ ID NO: 141) are targeted to human HIF-1α ASOs; ISIS222035 (SEQ ID NO: 244) is targeted to human HIF-2α; and ISIS 129688(TTCGCGGCTGGATTCAG; SEQ ID NO: 442) is an unrelated control. 10/35 is anequal mixture of ISIS 175510 and 222035 (HIF1● and HIF2● inhibitoryoligonucleotides). ISIS 97/35 is an equal mixture of ISIS 298697 andISIS 222035 (HIF1● and HIF2● inhibitory oligonucleotides).

TABLE 12 HIF1α and HIF2α play a major role in the induction of VEGF byhypoxia in HeLa cells Oligo- Relative nucleotide Normoxia VEGF mRNAexpression after treatment and or with oligonucleotide at concentrationsshown: conditions: Hypoxia 0 6.25 nM 25 nM 100 nM 200 nM 129688 N 100119 100 85 93 129688 H 284 283 289 234 209 175510 N 100 95 132 110 113175510 H 284 249 157 113 106 298697 N 100 84 105 93 93 298697 H 284 211144 106 108 222035 N 100 111 114 92 67 222035 H 284 260 209 144 77 10/35N 100 94 97 76 58 10/35 H 284 214 104 74 70 97/35 N 100 106 80 65 5697/35 H 284 207 108 73 60In this experiment all oligonucleotides except for the control (129688)interfered with induction of VEGF by hypoxia in HeLa cells. Thus bothHIF1α and HIF2α play a major role in the induction of VEGF by hypoxia inHeLa cells. Because the relative role of HIF1α and HIF2α in hypoxicinduction depends both on cell type and by induced gene (e.g., VEGF vsEpo), it is believed to be preferred to target both HIF1α and HIF2α forantisense inhibition. This may be achieved by a single cross-HIFantisense compound (such as ISIS 298697) or by a combination of one ormore antisense compounds targeted to HIF1α and one or more antisensecompounds targeted to HIF2α. Compounds administered in combination maybe given simultaneously or sequentially.

Example 29 Designing and Testing HIF1●/HIF2● Cross-Reacting AntisenseCompounds

The human HIF1α and HIF2α target sequences were compared for regions ofidentity but none were found to be as long as 20 nucleotides. However,based on the somewhat limited sequence homology between the human HIF1αand HIF2α target sequences, a series of antisense sequences weredesigned which were perfectly complementary to either HIF1α or HIF2α andwhich had no more than 4 mismatches to the other HIFα. These compoundsare shown in Table 13. The primary target sequence (perfectcomplementarity) is shown in the “target” column and the number ofmismatches against the other HIF is shown in subsequent columns. “Targetsite” refers to position on the primary target sequence.

TABLE 13 HIF1α/HIF2α crossreacting antisense sequences SEQ #Mis- # Mis-ISIS ID match vs matches vs Target NO OLIGO_SEQ NO Target HIF1α HIF2αsite HIF1α~EC50 HIF2α~EC50 129688 TTCGCGGCTGGACGATTCAG 442 Control330460 CCTCATGGTCGCAGGGATGA 443 HIF2α 2 554 (G-A, G-U) 330462TCTCCTCATGGTCGCAGGGA 444 HIF2α 3 557 (G-A, G-U, C-A) 222013CGAATCTCCTCATGGTCGCA 233 HIF2α 4 561 (G-U, C-A, A-G, G-A) 298697TCCTCATGGTCACATGGATG 141 HIF1α 2 673 5 30 (A-C, T-C) 330447TCATGGTCACATGGATGAGT 445 HIF1α 2 670 8 50 A-C, T-C) 330449CCTCATGGTCACATGGATGA 446 HIF1α 2 672 5 30 A-C, T-C) 330448CTCATGGTCACATGGATGAG 447 HIF1α 2 671 (A-C, T-C) 330452ATTTCCTCATGGTCACATGG 448 HIF1α 3 676 (A-C, T-C, G-T) 330470AAACCCTCCAAGGCTTTCAG 449 HIF2α 2 423 45 20 (G-U, C-U) 326743TCCTCATGGTCGCAGGGATG 450 HIF2α 2 555 40 10 G-A, G-U)Thus it is possible to inhibit both HIF1α and HIF2α with a singlecrossreacting oligonucleotide, although the relative antisense efficacyis not equal for the two forms because of imperfect homology to one HIFαor the other.

Example 30 Crossr Acting HIF1α/HIF2α Antisense Compounds Containing“Universal” Bases

In order to try to get antisense compounds that were highly potentagainst both HIF1α and HIF2α targets, the nucleobases at the sites ofthe mismatches against one or the other HIF were replaced with the“universal bases” inosine or 3′nitro-pyrrole. Inosine has the ability topair with G, U or C. If there was an A at the particular position ofeither of the sequences, we used 3-nitropyrrole. This is a base thatdoes not have binding affinity to any of the bases, but also does notcause steric hindrance of the duplex. These oligos were screened andfound to be active against both targets with an intermediate potency.This is shown in Table 14. In the table, “I” indicates inosine and “P”indicates 3-nitropyrrole.

TABLE 14 HIF1α/HIF2α crossreacting antisense compounds containinguniversal bases SEQ # Mis- # Mis- ID match vs match vs Target ISIS NOOLIGO_SEQ NO Target HIF1α HIF2α site HIF1α~EC50 HIF2α~EC50 326743TCCTCATGGTCGCAGGGATG 450 HIF2α 2 555 40 10 (G-A, G-U) 298697TCCTCATGGTCACATGGATG 141 HIF1α 2 673 5 30 (A-C, T-C) 330449CCTCATGGTCAPCATGGATGA 446 HIF1α 2 672 5 30 (A-C, T-C) 337223TCCTCATGGTCICAPGGATG 451 HIF1α 2 2 673 25 15 and (I-T, P-A) (I-C, P-C)HIF2α 337224 CCTCATGGTCICAPGGATGA 452 HIF1α 2 2 672 25 15 and (I-T, P-A)(I-C, P-C) HIF2αIntroduction of universal bases into the antisense compounds at the siteof mismatches resulted in a more equal inhibitory effect for both HIF1αand HIF2α.

Example 31 Tube Formation Assay to Determine Effect of HIF1α and HIF2αAntisense Inhibitors on Angiogenesis

Angiogenesis is stimulated by numerous factors that promote interactionof endothelial cells with each other and with extracellular matrixmolecules, resulting in the formation of capillary tubes. This processcan be reproduced in tissue culture by the formation of tube-likestructures by endothelial cells. Loss of tube formation in vitro hasbeen correlated with the inhibition of angiogenesis in vivo (Carmelietet al., (2000) Nature 407:249–257; and Zhang et al., (2002) CancerResearch 62:2034–42), which supports the use of in vitro tube formationas an endpoint for angiogenesis.

Angiogenesis, or neovascularization, is the formation of new capillariesfrom existing blood vessels. In adult organisms this process istypically controlled and short-lived, for example in wound repair andregeneration. However, aberrant capillary growth can occur and thisuncontrolled growth plays a causal and/or supportive role in manypathologic conditions such as tumor growth and metastasis. In thecontext of this invention “aberrant angiogenesis” refers to unwanted oruncontrolled angiogenesis. Angiogenesis inhibitors are being evaluatedfor use as antitumor drugs. Other diseases and conditions associatedwith angiogenesis include arthritis, cardiovascular diseases, skinconditions, and aberrant wound healing. Aberrant angiogenesis can alsooccur in the eye, causing loss of vision. Examples of ocular conditionsinvolving aberrant angiogenesis include macular degeneration, diabeticretinopathy and retinopathy of prematurity.

The tube formation assay is performed using an in vitro AngiogenesisAssay Kit (Chemicon International, Temecula, Calif.), or growth factorreduced Matrigel (BD Biosciences, Bedford, Mass.). HUVECs were plated at4000 cells/well in 96-well plates. One day later, cells were transfectedwith antisense and control oligonucleotides according to standardpublished procedures (Monia et al., (1993) J Biol Chem. 1993 Jul.5;268(19):14514–22) using 75 nM oligonucleotide in lipofectin (Gibco,Grand Island, N.Y.). Approximately fifty hours post-transfection, cellswere transferred to 96-well plates coated with ECMatrix™ (ChemiconInternational) or growth factor depleted Matrigel. Under theseconditions, untreated HUVECs form tube-like structures. After anovernight incubation at 37° C., treated and untreated cells wereinspected by light microscopy. Individual wells were assigned discretescores from 1 to 5 depending on the extent of tube formation. A score of1 refers to a well with no tube formation while a score of 5 is given towells where all cells are forming an extensive tubular network.

Table 15 Effect of HIF1α and HIF2α Antisense Oligonucleotides onAngiogenic Tube Formation

ISIS 29848 (NNNNNNNNNNNNNNNNNNNN; SEQ ID NO: 453) is a controloligonucleotide containing an equal mixture of the bases A, C, G and Tat every position. ISIS 298695 (SEQ ID NO: 139) and ISIS 298750 (Seq;SEQ ID NO: 193) are targeted to HIF1α; ISIS 330447 (Seq; SEQ ID NO: 445)is a cross-HIF oligonucleotide having perfect complementarity to HIF1αtarget and imperfect complementarity (and thus less inhibitory effect)for HIF2α; ISIS 222035 (SEQ ID NO: 244) and 222025 (SEQ ID NO: 239) aretargeted to HIF2α and ISIS 326743 is a cross-HIF oligonucleotide havingperfect complementarity to HIF2α target and imperfect complementarity(and thus less inhibitory effect) for HIF1α.

ISIS # Target 0 10 nM 35 nM 75 nM  29848 control 5 5 4.75 4.375 298695HIF1α 5 5 5 3.75 298750 HIF1α 5 5 4.75 3.25 330447 HIF 1α/ 5 5 4.25 3 2α222035 HIF2α 5 5 3.75 1.75 222025 HIF2α 5 5 3.5 1.75 326743 HIF2α/ 5 54.75 5 1αAs calculated from the assigned discrete scores, it is apparent thatHUVEC tube formation is inhibited by reduction of HIF2α and HIF1α,singly or in combination.

Example 32 Inhibition of HIF1α Expression In Vivo

C57B1/6 mice are maintained on a standard rodent diet and are used ascontrol (lean) animals. Seven-week old male C57B1/6 mice are injectedsubcutaneously with oligonucleotides at a dose of 25 mg/kg two times perweek for 4 weeks. Saline-injected animals serve as a control. After thetreatment period, mice are sacrificed and target levels are evaluated inliver, using RNA isolation and target mRNA expression level quantitation(RT-PCR) as described in other examples herein.

Oligonucleotides used in this experiment were ISIS 298695 (SEQ ID NO:139), ISIS 298697 (SEQ ID NO: 141), and ISIS 298750, (SEQ ID NO: 193),all targeted to mouse HIF1● and crossreactive to human HIF1● ISIS 141923(CCTTCCCTGAAGGTTCCTCC; SEQ ID NO: 454) is an unrelated negative controloligonucleotide. Results are shown in Table 16.

TABLE 16 Antisense inhibition of HIF1• expression in mouse liver byantisense to HIF1• ISIS # % inhib. of HIF1• Saline 0 ISIS 298695 76 ISIS298697 70 ISIS 298750 74 ISIS 141923 0 (control)

The effect of inhibiting HIF1α on levels of VEGF and GLUT1 in mouseliver was also determined. These are both hypoxia-inducible targets.Results are shown in Table 17 and 18.

TABLE 17 Effect of Antisense inhibition of HIF1• on VEGF expression inmouse liver ISIS # % inhib. of VEGF Saline 0 ISIS 298695 12 ISIS 2986974 ISIS 298750 0 ISIS 141923 0 (control)

TABLE 18 Effect of antisense inhibition of HIF1• on GLUT1 expression inmouse liver ISIS # % inhib. of VEGF Saline 0 ISIS 298695 0 ISIS 29869715 ISIS 298750 0 ISIS 141923 22 (control)

Example 33 Antisense Inhibition of HIF1α in a Mouse Model ofHepatocellular Carcinoma (HCC)

An HCC mouse model (C57BL/6-TgN(CRP-TagSV40)60-4, Taconic, GermantownN.Y.) for hepatocellular carcinoma was used in which transgenic malemice express SV40 T-antigen (Tag) in their livers, which leads tospontaneous development of well-differentiated hepatocellular carcinoma(HCC) carcinomas (Ruther et al., 1993, Oncogene 8, 87–93). HCC mice weretreated with ISIS 298695, ISIS 298697 or ISIS 298750, all targeted toHIF1● or with an unrelated control oligonucleotide. HCC and wild typemice were also treated with saline alone. Results are shown in Table 19.

TABLE 19 Antisense inhibition of HIF1• in HCC mouse liver ISIS # SEQ IDNO % inhib. of HIF1• Saline 0 ISIS 298695 139 43 ISIS 298697 141 33 ISIS298750 193 40 ISIS 141923 454 11 (control) C57BL6/saline 43The effect of HIF1● inhibition on GLUT1 expression in HCC mice was alsoevaluated. Results are shown in Table 20.

TABLE 20 Effect of antisense inhibition of HIF1• on GLUT1 levels in HCCmouse liver ISIS # SEQ ID NO % inhib. of GLUT1 Saline 0 ISIS 298695 1390 ISIS 298697 141 0 ISIS 298750 193 13 ISIS 141923 454 18 (control)C57BL6/saline 2

Example 34 Inhibition of HIF2α Expression in Tumor Cells by Wild-Typep53 Under Hypoxia in T47D Tumor Cells

T47D breast adenocarcinoma cells were obtained from the American TypeCulture Collection (ATCC) (Manassas, Va.). Cells were cultured in GibcoDMEM High glucose media supplemented with 10% FBS. p53 is a tumorsuppressor gene product which is inactive or aberrant in approximately50% of human tumors. T47D cells are p53 null, i.e. they contain inactivemutant p53. These cells express high levels of HIF2● even in normoxicconditions. Hypoxia or CoCl₂ induces even higher levels of HIF2●expression. In contrast, T47D cells which have been transfected with aplasmid expressing p53, thus restoring p53 function in these cells,express extremely low levels of HIF2●, even in hypoxic conditions or inCoCl₂ simulation of hypoxia. This increase in HIF2● in cells withaberrant p53 is believed to be a novel observation and is believed toindicate a link between p53 and the HIF pathway.

Example 35 Effects of Antisense Inhibition of HIF1α and/or HIF2α onCancer Cell Proliferation Under Hypoxia/Glucose Deprivation

PC-3 human prostate cancer cells were cultured as described in previousexamples. Cells were electroporated with oligonucleotide atconcentrations described below and grown for 16 hours at normoxia and0.45 g/l glucose. The medium was then replaced with either glucose (4.5g/l glucose) or low-glucose medium (no added glucose) and cells werethen kept at hypoxia (1% O₂) or normoxia (21% O₂) for another 48 hours.The effect of antisense treatment on cell proliferation was measured.Oligonucleotides were ISIS 129688 (unrelated control), ISIS 175510(HIF1α), ISIS 222035 (HIF2α) and ISIS 298697 (HIF1α with somecrossreactivity to HIF2α). Results are shown in the tables below, onetable for each culture condition.

TABLE 21 Effect of HIF antisense on proliferation of PC-3 cancer cellsNormoxia/Glucose Cell proliferation as percent of saline control ISIS #0 nM 10 nM 20 nM SEQ ID NO 129688 100 103 103 442 175510 100 126 93 47222035 100 130 116 244 298697 100 118 86 141

TABLE 22 Effect of HIF antisense on proliferation of PC-3 cancer cellsHypoxia/Glucose Cell proliferation as percent of saline control ISIS # 0nM 10 nM 20 nM SEQ ID NO 129688 100 104 99 442 175510 100 113 105 47222035 100 106 91 244 298697 100 113 83 141

TABLE 23 Effect of HIF antisense on proliferation of PC-3 cancer cellsNormoxia/Low Glucose Cell proliferation as percent of saline controlISIS # 0 nM 10 nM 20 nM SEQ ID NO 129688 100 107 105 442 175510 100 9689 47 222035 100 91 68 244 298697 100 91 88 141

TABLE 24 Effect of HIF antisense on proliferation of PC-3 cancer cellsHypoxia/Low Glucose Cell proliferation as percent of saline control ISIS# 0 nM 10 nM 20 nM SEQ ID NO 129688 100 105 103 442 175510 100 90 85 47222035 100 88 80 244 298697 100 88 61 141

Example 36 Effect of Antisense Inhibitors of HIFs on Human Tumor CellXenografts in Mice

Nude mice are injected in the flank with approximately 10⁶ U87-MG humanglioblastoma cells. Mice are dosed with antisense compound beginning theday after tumor inoculation and continuing every other day.Oligonucleotides used are ISIS 129688 (unrelated control), ISIS 175510(HIF1α), ISIS 222035 (HIF2α) and ISIS 298697 (HIF1α with somecrossreactivity to HIF2α). Tumor volume is measured every few daysbeginning 10 days after inoculation.

Similar xenograft studies are performed with MDA-MB231 human breastcancer cells, which are p53-deficient. Nude mice are injected in theflank with approximately 10⁶ MDA-MB231 human breast cancer cells. Miceare dosed with antisense compound beginning the day after tumorinoculation and continuing every other day. Oligonucleotides used areISIS 129688 (unrelated control), ISIS 175510 (HIF1α), ISIS 222035(HIF2α) and ISIS 298697 (HIF1α with some crossreactivity to HIF2α).Tumor volume is measured every few days beginning 10 days afterinoculation.

Example 37 Effect of Antisense Inhibitors of HIFs on AngiogenicConditions in the Eye

It is believed that antisense inhibitors of HIF2α and possibly HIF1αwill be useful in treatment of angiogenic conditions, because of theireffect on endothelial tube formation in an in vitro model forangiogenesis (see previous examples).

A pig model of ocular neovascularization, the branch retinal veinocclusion (BVO) model, is used to study ocular neovascularization. Malefarm pigs (8–10 kg) are subjected to branch retinal vein occlusions(BVO) by laser treatment in both eyes. The extent of BVO is determinedby indirect opthalmoscopy after a 2 week period. Intravitreousinjections (10 ●M) of ISIS 129688 (unrelated control), ISIS 175510(HIF1α), ISIS 222035 (HIF2α) or ISIS 298697 (HIF1α with somecrossreactivity to HIF2α) are started on the day of BVO induction andare repeated at weeks 2, 6, and 10 after BVO (Right eye—vehicle, Lefteye—antisense molecule). Stereo fundus photography and fluoresceinangiography are performed at baseline BVO and at weeks 1, 6 and 12following intravitreous injections to measure the neovascular response.In addition capillary gel electrophoresis analysis of the eye sectionscontaining sclera, choroid, and the retina are performed to determineantisense concentrations, and gross and microscopic evaluations areperformed to determine eye histopathology.

1. An antisense oligonucleotide 15 to 30 nucleobases in length targetedto a nucleic acid molecule encoding HIF1α (SEQ ID NO: 133), wherein saidcompound comprises at least 8 consecutive nucleobases of SEQ ID NO:446.2. The antisense oligonucleotide of claim 1 wherein said antisenseoligonucleotide is a DNA oligonucleotide.
 3. The antisenseoligonucleotide of claim 1 wherein said antisense oligonucleotide is anRNA oligonucleotide.
 4. The antisense oligonucleotide of claim 1 whereinsaid antisense oligonucleotide is a chimeric oligonucleotide.
 5. Theantisense oligonucleotide of claim 1 comprising at least one modifiedinternucleoside linkage, sugar moiety, or nucleobase.
 6. The antisenseoligonucleotide of claim 1 comprising at least one 2′-O-methoxyethylsugar moiety.
 7. The antisense oligonucleotide of claim 1 comprising atleast one phosphorothioate internucleoside linkage.
 8. The antisenseoligonucleotide of claim 1 comprising at least one 5-methylcytosine. 9.A method of inhibiting the expression of HIF1α in a cell in vitrocomprising contacting said cell with the antisense oligonucleotide ofclaim
 1. 10. A kit or assay device comprising the antisenseoligonucleotide of claim
 1. 11. A composition comprising the antisenseoligonucleotide of claim 1 in a pharmaceutically acceptable carrier. 12.An antisense oligonucleotide with a nucleotide sequence consisting ofSEQ ID NO:
 446. 13. The antisense oligonucleotide of claim 1 having 100%complementarity with the nucleic acid molecule encoding HIF1α.
 14. Anantisense oligonucleotide 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25nucleobases in length targeted to a nucleic acid molecule encodingHIF1-alpha (SEQ ID NO: 133), wherein said antisense oligonucleotide hasat least 80% identity with SEQ ID NO:
 446. 15. The antisenseoligonucleotide of claim 14 which is 18, 19, 20, 21 or 22 nucleobases inlength and has at least 90% identity with SEQ ID NO:
 446. 16. Theantisense oligonucleotide of claim 15 which is 19, 20 or 21 nucleobasesin length and has at least 95% identity with SEQ ID NO:
 446. 17. Theantisense oligonacleotide of claim 12 comprising a central region of ten2′-deoxynucleotides which is flanked on each side by five2′-O-methoxyethyl nucleotides, wherein the internucleoside linkages ofsaid oligonucleotide are phosphorothioate throughout the oligonucleotideand the cytidine residues are 5-niethylcytidines.
 18. A pharmaceuticalcomposition comprising the antisense oligonucleotide of claim 17.